scholarly journals ERK5 is targeted to myocyte enhancer factor 2A (MEF2A) through a MAPK docking motif

2004 ◽  
Vol 381 (3) ◽  
pp. 693-699 ◽  
Author(s):  
Dalia BARSYTE-LOVEJOY ◽  
Alex GALANIS ◽  
Anne CLANCY ◽  
Andrew D. SHARROCKS
Keyword(s):  
Catalytic Domain ◽  
Mutational Analysis ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Substrate ◽  
Myocyte Enhancer Factor ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Enhancer Factor

One critical component in determining the specificity, and efficiency of MAPK (mitogen-activated protein kinase) substrate phophorylation is the presence of distinct docking domains in the substrate proteins. Docking domains have been shown to be important for the activities of members of the ERK (extracellular-signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 subfamilies of MAPKs towards their substrates. Here, we demonstrate that docking domains also play an important role in ERK5-mediated substrate phosphorylation. The presence of a docking domain promotes both phosphorylation of myocyte enhancer factor, MEF2A, in vitro and its activation in vivo by ERK5. Mutational analysis of the MEF2A docking domain demonstrates that the specificity determinants for ERK5 are similar to those observed with members of the p38 subfamily. A docking domain recognized by ERK5 can direct ERK5 to activate heterologous substrates. Deletion analysis demonstrates that as with other MAPKs, it is the catalytic domain of ERK5 that recognizes the docking domain. Our data therefore extend previous observations on other MAPKs and demonstrate that the requirement for specific docking domains in promoting MAPK action towards substrates is a general property of MAPKs.

scholarly journals Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation

2005 ◽  
Vol 25 (2) ◽  
pp. 854-864 ◽  
Author(s):  
Sandrine Marchetti ◽  
Clotilde Gimond ◽  
Jean-Claude Chambard ◽  
Thomas Touboul ◽  
Danièle Roux ◽  
...  
Keyword(s):  
Map Kinases ◽  
Fluorescent Protein ◽  
Proteasomal Degradation ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Double Mutation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.

scholarly journals Chinese herbal formulas for postmenopausal osteoporosis: A review of preclinical evidence on animal studies and molecular mechanism

2018 ◽  
Vol 01 (02) ◽  
pp. 75-83
Author(s):  
Jing Lin ◽  
Tian Lv ◽  
Fubo Tian ◽  
Yan Wang ◽  
Mingyan Wang ◽  
...  
Keyword(s):  
Postmenopausal Osteoporosis ◽  
Animal Studies ◽  
Mitogen Activated Protein Kinase ◽  
Chinese Herbal ◽  
Morphogenetic Protein ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Underlying Mechanisms

Whether Chinese herbal formulas are effective in treatment of postmenopausal osteoporosis remains unclear. The aim of this study is to explore the experimental evidence of both in vitro and in vivo preclinical studies using Chinese herbal formulas in postmenopausal osteoporosis. Searches were applied to various databases with relevant keywords. Original in vivo and in vitro studies using Chinese herbal formulas to treat postmenopausal osteoporosis, and with full text available, were included. Er-Xian Decoction, Bu-Shen-Ning-Xin Decoction, Qing E Formula, Liuwei Dihuang Wan, and Xian-Ling-Gu-Bao Decoction, the most commonly studied formulas, were selected from the pool of Chinese medicine. The preclinical data indicated the potential use of Chinese herbal formulas in postmenopausal osteoporosis. The underlying mechanisms included bone morphogenetic protein (BMP), Wnt/[Formula: see text]-catenin, extracellular-signal-regulated kinase/c-Jun [Formula: see text] terminal kinase/mitogen-activated protein kinase (ERK/JNK/MAPK), estrogen receptor (ER), and osteoprotegerin/receptor activator of nuclear factor [Formula: see text]B ligand (OPG/RANKL) signaling pathways. This study demonstrated the anti-osteoporotic effect of Chinese herbal formulas targeting different pathways in bone metabolism. Further study with adequate sample size and follow-up time, appropriate controls, and optimal blinding is required.

scholarly journals Optimizing photoswitchable MEK

2019 ◽  
Vol 116 (51) ◽  
pp. 25756-25763 ◽  
Author(s):  
Aleena L. Patel ◽  
Eyan Yeung ◽  
Sarah E. McGuire ◽  
Andrew Y. Wu ◽  
Jared E. Toettcher ◽  
...  
Keyword(s):  
Mitogen Activated Protein Kinase ◽  
Kinase Activation ◽  
Erk Activation ◽  
Signaling Systems ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Morphogenetic Event ◽  
Zebrafish Embryogenesis

Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in bothDrosophilaand zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.

scholarly journals An analysis of the phosphorylation and activation of extracellular-signal-regulated protein kinase 5 (ERK5) by mitogen-activated protein kinase kinase 5 (MKK5) in vitro

2003 ◽  
Vol 372 (2) ◽  
pp. 567-575 ◽  
Author(s):  
Nimesh MODY ◽  
David G. CAMPBELL ◽  
Nick MORRICE ◽  
Mark PEGGIE ◽  
Philip COHEN
Keyword(s):  
Protein Kinase ◽  
Catalytic Domain ◽  
Similar Rate ◽  
Mitogen Activated Protein Kinase ◽  
Glutathione S Transferase ◽  
293 Cells ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Protein Kinase Kinase

MKK5 expressed as a glutathione S-transferase fusion protein in human embryonic kidney 293 cells activated full-length extracellular-signal-regulated protein kinase (ERK)5 (ERK5wt) as well as the isolated catalytic domain (ERK5cat) in vitro. Activation was accompanied by the phosphorylation of Thr219 and Tyr221, the former residue being phosphorylated preferentially. ERK5cat phosphorylated at Thr219, but not Tyr221, possessed 10% of the activity of the doubly phosphorylated protein towards myelin basic protein, whereas ERK5cat phosphorylated at Tyr221 alone was much less active. Activated ERK5 phosphorylated itself at a number of residues, including Thr28, Ser421, Ser433, Ser496, Ser731 and Thr733. ERK5 phosphorylated at Thr219, but not Tyr221, phosphorylated itself at a similar rate to ERK5 phosphorylated at both Thr219 and Tyr221. Activated ERK5 also phosphorylated mitogen-activated protein kinase kinase 5 (MKK5) extensively at Ser129, Ser137, Ser142 and Ser149, which are located within the region in MKK5 that is thought to interact with ERK5.

scholarly journals Mutational Analysis of STE5 in the Yeast Saccharomyces cerevisiae: Application of a Differential Interaction Trap Assay for Examining Protein-Protein Interactions

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 479-492 ◽  
Author(s):  
Carla Inouye ◽  
Namrita Dhillon ◽  
Tim Durfee ◽  
Patricia C Zambryski ◽  
Jeremy Thorner
Keyword(s):  
Protein Interactions ◽  
Mutational Analysis ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Missense Mutations ◽  
Protein Protein Interactions ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mitogen Activated Protein

Ste5 is essential for the yeast mating pheromone response pathway and is thought to function as a scaffold that organizes the components of the mitogen-activated protein kinase (MAPK) cascade. A new method was developed to isolate missense mutations in Ste5 that differentially affect the ability of Ste5 to interact with either of two MAPK cascade constituents, the MEKK (Ste11) and the MEK (Ste7). Mutations that affect association with Ste7 or with Ste11 delineate discrete regions of Ste5 that are critical for each interaction. Co-immunoprecipitation analysis, examining the binding in vitro of Ste5 to Ste11, Ste7, Ste4 (G protein, β subunit), and Fus3 (MAPK), confirmed that each mutation specifically affects the interaction of Ste5 with only one protein. When expressed in a ste5Δ cell, mutant Ste5 proteins that are defective in their ability to interact with either Ste11 or Ste7 result in a markedly reduced mating proficiency. One mutation that clearly weakened (but did not eliminate) interaction of Ste5 with Ste7 permitted mating at wild-type efficiency, indicating that an efficacious signal is generated even when Ste5 associates with only a small fraction of (or only transiently with) Ste7. Ste5 mutants defective in association with Ste11 or Ste7 showed strong interallelic complementation when co-expressed, suggesting that the functional form of Ste5 in vivo is an oligomer.

Regulation and distribution of MAdCAM-1 in endothelial cells in vitro

2001 ◽  
Vol 281 (4) ◽  
pp. C1096-C1105 ◽  
Author(s):  
Tadayuki Oshima ◽  
Kevin P. Pavlick ◽  
F. Stephen Laroux ◽  
S. Kris Verma ◽  
Paul Jordan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Extracellular Signal ◽  
Tnf Α ◽  
Mitogen Activated Protein ◽  
Cell Adhesion Molecule 1

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a 60-kDa endothelial cell adhesion glycoprotein that regulates lymphocyte trafficking to Peyer's patches and lymph nodes. Although it is widely agreed that MAdCAM-1 induction is involved in chronic gut inflammation, few studies have investigated regulation of MAdCAM-1 expression. We used two endothelial lines [bEND.3 (brain) and SVEC (high endothelium)] to study the signal paths that regulate MAdCAM-1 expression in response to tumor necrosis factor (TNF)-α using RT-PCR, blotting, adhesion, and immunofluorescence. TNF-α induced both MAdCAM-1 mRNA and protein in a dose- and time-dependent manner. This induction was tyrosine kinase (TK), p42/44, p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB/poly-ADP ribose polymerase (PARP) dependent. Because MAdCAM-1 is regulated via MAPKs, we examined mitogen/extracellular signal-regulated kinase (MEK)-1/2 activation in SVEC. We found that MEK-1/2 is activated by TNF-α within minutes and is dependent on TK and p42/44 MAPKs. Similarly, TNF-α activated NF-κB through TK, p42/44, p38 MAPKs, and PARP pathways in SVEC cells. MAdCAM-1 was also shown to be frequently distributed to endothelial junctions both in vitro and in vivo. Cytokines like TNF-α stimulate MAdCAM-1 in high endothelium via TK, p38, p42/22 MAPKs, and NF-κB/PARP. MAdCAM-1 expression requires NF-κB translocation through both direct p42/44 and indirect p38 MAPK pathways in high endothelial cells.

scholarly journals Actions of Brain-Derived Neurotrophin Factor in the Neurogenesis and Neuronal Function, and Its Involvement in the Pathophysiology of Brain Diseases

2018 ◽  
Vol 19 (11) ◽  
pp. 3650 ◽  
Author(s):  
Tadahiro Numakawa ◽  
Haruki Odaka ◽  
Naoki Adachi
Keyword(s):  
Synaptic Function ◽  
Mitogen Activated Protein Kinase ◽  
Brain Diseases ◽  
Neuronal Function ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
High Affinity Receptor ◽  
The Central Nervous System

It is well known that brain-derived neurotrophic factor, BDNF, has an important role in a variety of neuronal aspects, such as differentiation, maturation, and synaptic function in the central nervous system (CNS). BDNF stimulates mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), phosphoinositide-3kinase (PI3K), and phospholipase C (PLC)-gamma pathways via activation of tropomyosin receptor kinase B (TrkB), a high affinity receptor for BDNF. Evidence has shown significant contributions of these signaling pathways in neurogenesis and synaptic plasticity in in vivo and in vitro experiments. Importantly, it has been demonstrated that dysfunction of the BDNF/TrkB system is involved in the onset of brain diseases, including neurodegenerative and psychiatric disorders. In this review, we discuss actions of BDNF and related signaling molecules on CNS neurons, and their contributions to the pathophysiology of brain diseases.

scholarly journals Identification of Novel In Vivo Raf-1 Phosphorylation Sites Mediating Positive Feedback Raf-1 Regulation by Extracellular Signal-regulated Kinase

2006 ◽  
Vol 17 (3) ◽  
pp. 1141-1153 ◽  
Author(s):  
Vitaly Balan ◽  
Deborah T. Leicht ◽  
Jun Zhu ◽  
Karina Balan ◽  
Alexander Kaplun ◽  
...  
Keyword(s):  
Positive Feedback ◽  
Critical Role ◽  
Mitogen Activated Protein Kinase ◽  
Phosphorylation Sites ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Phosphopeptide Mapping

The Ras–Raf–mitogen-activated protein kinase cascade is a key growth-signaling pathway, which uncontrolled activation results in transformation. Although the exact mechanisms underlying Raf-1 regulation remain incompletely understood, phosphorylation has been proposed to play a critical role in this regulation. We report here three novel epidermal growth factor-induced in vivo Raf-1 phosphorylation sites that mediate positive feedback Raf-1 regulation. Using mass spectrometry, we identified Raf-1 phosphorylation on three SP motif sites: S289/S296/S301 and confirmed their identity using two-dimensional-phosphopeptide mapping and phosphospecific antibodies. These sites were phosphorylated by extracellular signal-regulated kinase (ERK)-1 in vitro, and their phosphorylation in vivo was dependent on endogenous ERK activity. Functionally, ERK-1 expression sustains Raf-1 activation in a manner dependent on Raf-1 phosphorylation on the identified sites, and S289/296/301A substitution markedly decreases the in vivo activity of Raf-1 S259A. Importantly, the ERK-phosphorylated Raf-1 pool has 4 times higher specific kinase activity than total Raf-1, and its phosphopeptide composition is similar to that of the general Raf-1 population, suggesting that the preexisting, phosphorylated Raf-1, representing the activatable Raf-1 pool, is the Raf-1 subpopulation targeted by ERK. Our study describes the identification of new in vivo Raf-1 phosphorylation sites targeted by ERK and provides a novel mechanism for a positive feedback Raf-1 regulation.

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