Identification ofVibrio choleraeType III Secretion System Effector Proteins

ABSTRACTAM-19226 is a pathogenic O39 serogroupVibrio choleraestrain that lacks the typical virulence factors for colonization (toxin-coregulated pilus [TCP]) and toxin production (cholera toxin [CT]) and instead encodes a type III secretion system (T3SS). The mechanism of pathogenesis is unknown, and few effector proteins have been identified. We therefore undertook a survey of the open reading frames (ORFs) within the ∼49.7-kb T3SS genomic island to identify potential effector proteins. We identified 15 ORFs for their ability to inhibit growth when expressed in yeast and then used a β-lactamase (TEM1) fusion reporter system to demonstrate that 11 proteins werebona fideeffectors translocated into HeLa cellsin vitroin a T3SS-dependent manner. One effector, which we named VopX (A33_1663), is conserved only inV. choleraeandVibrio parahaemolyticusT3SS-positive strains and has not been previously studied. AvopXdeletion reduces the ability of strain AM-19226 to colonizein vivo, and the bile-induced expression of avopX-lacZtranscriptional fusionin vitrois regulated by the T3SS-encoded transcriptional regulators VttRAand VttRB. AnRLM1yeast deletion strain rescued the growth inhibition induced by VopX expression, suggesting that VopX interacts with components of the cell wall integrity mitogen-activated protein kinase (MAPK) pathway. The collective results show that theV. choleraeT3SS encodes multiple effector proteins, one of which likely has novel activities that contribute to disease via interference with eukaryotic signaling pathways.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.200610046 ◽

2007 ◽

Vol 176 (5) ◽

pp. 709-718 ◽

Cited By ~ 337

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.25.2.854-864.2005 ◽

2005 ◽

Vol 25 (2) ◽

pp. 854-864 ◽

Cited By ~ 90

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.

Download Full-text

p120 catenin induces opposing effects on tumor cell growth depending on E-cadherin expression

The Journal of Cell Biology ◽

10.1083/jcb.200805113 ◽

2008 ◽

Vol 183 (4) ◽

pp. 737-749 ◽

Cited By ~ 63

Author(s):

Edwin Soto ◽

Masahiro Yanagisawa ◽

Laura A. Marlow ◽

John A. Copland ◽

Edith A. Perez ◽

...

Keyword(s):

Cell Growth ◽

Tumor Cell ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

Tumor Cell Growth ◽

P120 Catenin ◽

Opposing Effects ◽

E Cadherin

p120 catenin regulates the activity of the Rho family guanosine triphosphatases (including RhoA and Rac1) in an adhesion-dependent manner. Through this action, p120 promotes a sessile cellular phenotype when associated with epithelial cadherin (E-cadherin) or a motile phenotype when associated with mesenchymal cadherins. In this study, we show that p120 also exerts significant and diametrically opposing effects on tumor cell growth depending on E-cadherin expression. Endogenous p120 acts to stabilize E-cadherin complexes and to actively promote the tumor-suppressive function of E-cadherin, potently inhibiting Ras activation. Upon E-cadherin loss during tumor progression, the negative regulation of Ras is relieved; under these conditions, endogenous p120 promotes transformed cell growth both in vitro and in vivo by activating a Rac1–mitogen-activated protein kinase signaling pathway normally activated by the adhesion of cells to the extracellular matrix. These data indicate that both E-cadherin and p120 are important regulators of tumor cell growth and imply roles for both proteins in chemoresistance and targeted therapeutics.

Download Full-text

Phosphorylation of High-Mobility Group Protein A2 by Nek2 Kinase during the First Meiotic Division in Mouse Spermatocytes

Molecular Biology of the Cell ◽

10.1091/mbc.e03-09-0638 ◽

2004 ◽

Vol 15 (3) ◽

pp. 1224-1232 ◽

Cited By ~ 55

Author(s):

Silvia Di Agostino ◽

Monica Fedele ◽

Paolo Chieffi ◽

Alfredo Fusco ◽

Pellegrino Rossi ◽

...

Keyword(s):

Mapk Pathway ◽

Chromatin Condensation ◽

High Mobility ◽

Mitogen Activated Protein Kinase ◽

Dependent Manner ◽

Correct Process ◽

High Mobility Group Protein ◽

Mitogen Activated Protein ◽

Pachytene Spermatocytes

The mitogen-activated protein kinase (MAPK) pathway is required for maintaining the chromatin condensed during the two meiotic divisions and to avoid a second round of DNA duplication. However, molecular targets of the MAPK pathway on chromatin have not yet been identified. Here, we show that the architectural chromatin protein HMGA2 is highly expressed in male meiotic cells. Furthermore, Nek2, a serine-threonine kinase activated by the MAPK pathway in mouse pachytene spermatocytes, directly interacts with HMGA2 in vitro and in mouse spermatocytes. The interaction does not depend on the activity of Nek2 and seems constitutive. On progression from pachytene to metaphase, Nek2 is activated and HMGA2 is phosphorylated in an MAPK-dependent manner. We also show that Nek2 phosphorylates in vitro HMGA2 and that this phosphorylation decreases the affinity of HMGA2 for DNA and might favor its release from the chromatin. Indeed, we find that most HMGA2 associates with chromatin in mouse pachytene spermatocytes, whereas it is excluded from the chromatin upon the G2/M progression. Because hmga2-/- mice are sterile and show a dramatic impairment of spermatogenesis, it is possible that the functional interaction between HMGA2 and Nek2 plays a crucial role in the correct process of chromatin condensation in meiosis.

Download Full-text

Phosphorylation of Argonaute 2 at serine-387 facilitates its localization to processing bodies

Biochemical Journal ◽

10.1042/bj20080599 ◽

2008 ◽

Vol 413 (3) ◽

pp. 429-436 ◽

Cited By ~ 134

Author(s):

Yan Zeng ◽

Heidi Sankala ◽

Xiaoxiao Zhang ◽

Paul R. Graves

Keyword(s):

Protein Kinase ◽

P38 Mapk ◽

Kinase Inhibitor ◽

Mapk Pathway ◽

Phosphorylation Site ◽

Mitogen Activated Protein Kinase ◽

Processing Bodies ◽

Mitogen Activated Protein

Ago (Argonaute) proteins are essential effectors of RNA-mediated gene silencing. To explore potential regulatory mechanisms for Ago proteins, we examined the phosphorylation of human Ago2. We identified serine-387 as the major Ago2 phosphorylation site in vivo. Phosphorylation of Ago2 at serine-387 was significantly induced by treatment with sodium arsenite or anisomycin, and arsenite-induced phosphorylation was inhibited by a p38 MAPK (mitogen-activated protein kinase) inhibitor, but not by inhibitors of JNK (c-Jun N-terminal kinase) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]. MAPKAPK2 (MAPK-activated protein kinase-2) phosphorylated bacterially expressed full-length human Ago2 at serine-387 in vitro, but not the S387A mutant. Finally, mutation of serine-387 to an alanine residue or treatment of cells with a p38 MAPK inhibitor reduced the localization of Ago2 to processing bodies. These results suggest a potential regulatory mechanism for RNA silencing acting through Ago2 serine-387 phosphorylation mediated by the p38 MAPK pathway.

Download Full-text

Taurine Activates BMP-2/Wnt3a-Mediated Osteoblast Differentiation and Mineralization via Akt and MAPK Signaling

Iranian Journal of Public Health ◽

10.18502/ijph.v48i11.3514 ◽

2020 ◽

Author(s):

Minsu PARK ◽

Hyeon Kyeong CHOI ◽

Jeung Hee AN

Keyword(s):

Protein Kinase ◽

Osteoblast Differentiation ◽

Integration Site ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Bone Morphogenetic Protein 2 ◽

Dependent Manner ◽

Ovx Rats

Background: We aimed to elucidate the preventive effects of taurine against osteopenia in ovariectomized (OVX) rats and the mechanisms by which taurine regulates osteoblastogenesis in vitro and in vivo. Methods: The effects of the taurine on human osteoblast MG-63 cell differentiation and osteoblastogenesis effect in OVX rat were examined Konkuk University in 2018 by evaluating osteoblast differentiation, and expression of osteoblast-specific factors by western blotting analysis. Results: Taurine supplementation significantly improved alkaline phosphatase (ALP) activity and mineralization in a concentration-dependent manner. Further, taurine induced the expression of osteogenic growth factors such as bone morphogenetic protein-2 (BMP-2), runt-related transcription factor 2 (RUNX2), small mothers against decapentaplegic 1/5/8 (SMAD1/5/8), wingless-type MMTV integration site family member 3A (Wnt3a), and collagen type 1 (COL-1) via mitogen-activated protein kinase (MAPK) and serine/threonine protein kinase (Akt). Moreover, the RUNX2 activity of the taurine-treated group was enhanced by proteinprotein interactions such as Wnt3a-induced p-AKT/RUNX2 and BMP-mediated SMADs/MAPK/RUNX2 interactions. Conclusion: Our in vitro and in vivo results suggested that taurine can be considered as a potential therapeutic candidate agent for preventing bone loss in postmenopausal osteoporosis.

Download Full-text

SPDR-1 HSP90 inhibition overcomes resistant to molecular targeted therapy in BRAFV600E mutant glioblastoma

Neuro-Oncology Advances ◽

10.1093/noajnl/vdab159.010 ◽

2021 ◽

Vol 3 (Supplement_6) ◽

pp. vi3-vi3

Author(s):

Jo Sasame ◽

Naoki Ikegaya ◽

Yohei Miyake ◽

Takahiro Hayashi ◽

Akito Oshima ◽

...

Keyword(s):

Targeted Therapy ◽

Molecular Targeted Therapy ◽

Mapk Pathway ◽

Mek Inhibitor ◽

Mtor Pathway ◽

Mitogen Activated Protein Kinase ◽

Hsp90 Inhibition ◽

Pdx Models

Abstract The BRAFV600E mutation results in the constitutive activation of downstream mitogen activated protein kinase (MAPK) pathway that promotes tumor growth. Recently, molecular targeted therapy using BRAF/MEK inhibitor has been reported for BRAFV600E mutant high-grade glioma, but the therapeutic effect is limited by the emergence of drug resistance. Herein, we established paired BRAFV600E mutant glioblastoma (GBM) patient-derived xenograft (PDX) models, which were derived from tumors at prior to and recurrence after molecular targeted therapy. These PDX models were found to extensively recapitulate the histology, genetic abnormalities, and even the clinical course of the patients. Furthermore, BRAF/MEK inhibitor gradually caused resistance in cell lines derived from specimens that initially responded to molecular targeted therapy. In this study, genomic and epigenomic changes had little effect on the resistance mechanism. On the other hand, we found that hyperactivation of the MAPK pathway through c-Raf and the AKT/mTOR pathway primarily caused resistance to molecular targeted therapy in BRAFV600E mutant GBM. Through a high throughput drug screening, we find that HSP90 inhibitor with BRAF/MEK inhibitor coordinately deactivates MAPK pathway and AKT/mTOR pathway, and mediates potent toxicity in vitro and in vivo in refractory and acquired resistant models. These findings support that this therapeutic approach can overcome the limitation of current molecular targeted therapy in BRAFV600E mutant GBM.

Download Full-text

Enhancing the Therapeutic Efficacy of KRASG12C Inhibitors in Lung Adenocarcinoma Cell Models by Cotargeting the MAPK Pathway or HSP90

Journal of Oncology ◽

10.1155/2021/2721466 ◽

2021 ◽

Vol 2021 ◽

pp. 1-13

Author(s):

Ying Liu ◽

Lei Wu ◽

Hong Lu ◽

En Wu ◽

Jun Ni ◽

...

Keyword(s):

Drug Resistance ◽

Lung Adenocarcinoma ◽

Tyrosine Kinases ◽

Tumor Regression ◽

Mapk Pathway ◽

Transcriptional Profiling ◽

Multidrug Resistant ◽

Mitogen Activated Protein Kinase

Background. KRASG12C inhibitors have shown promising efficacy in early clinical trials, but drug resistance compromises their long-term benefits. Therefore, it is critical to understand the mechanisms of drug resistance and to design appropriate combinatory treatments to improve efficacy. Methods. To understand the comprehensive mechanisms of drug resistance, we treated lung cancer cells with KRASG12C inhibitors for different periods and performed transcriptional profiling and signaling analysis to identify critical factors and pathways that drive drug tolerance and resistance. We also evaluated several drug combinations in vitro and in vivo to identify potentially effective therapeutics. Results. We found that the feedback activation of multiple receptor tyrosine kinases (RTKs) may have cooperatively induced intrinsic and adaptive resistance to KRASG12C inhibitors. Notably, continuous KRAS inhibition induced a multidrug-resistant phenotype, implying that upfront combinatory treatment might be required to treat this group of patients. We also demonstrated that concurrently targeting multiple nodes in the RTK/RAS/RAF/MEK/ERK axis improved the efficacy of KRASG12C inhibitors, mainly by suppressing the reactivation of the mitogen-activated protein kinase (MAPK) pathway. Moreover, the combined use of HSP90 and KRASG12C inhibitors effectively induced tumor regression in lung adenocarcinoma models in vitro and in vivo. Conclusion. Together, our findings revealed mechanisms underlying KRASG12C inhibitors resistance and provided novel candidate combinatory strategies to improve their anticancer activity.

Download Full-text

MEK-inhibitor-mediated rescue of skeletal myopathy caused by activating Hras mutation in a Costello syndrome mouse model

Disease Models & Mechanisms ◽

10.1242/dmm.049166 ◽

2021 ◽

Author(s):

William E. Tidyman ◽

Alice F. Goodwin ◽

Yoshiko Maeda ◽

Ophir D. Klein ◽

Katherine A. Rauen

Keyword(s):

Mouse Model ◽

Mapk Pathway ◽

Mek Inhibitor ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Costello Syndrome ◽

Skeletal Myopathy ◽

Mitogen Activated Protein

Costello syndrome (CS) is a congenital disorder caused by heterozygous activating germline HRAS mutations in the canonical Ras/mitogen-activated protein kinase (Ras/MAPK) pathway. CS is one of the RASopathies, a large group of syndromes due to mutations within various components of the Ras/MAPK pathway. An important part of the phenotype that greatly impacts quality of life is hypotonia. To gain a better understanding of the mechanisms underlying hypotonia in CS, a mouse model with an activating HrasG12V allele was utilized. We identified a skeletal myopathy that was due in part to an inhibition of embryonic myogenesis and myofiber formation, resulting in a reduction of myofiber size and number that led to reduced muscle mass and strength. In addition to hyperactivation of the Ras/MAPK and PI3K/AKT pathways, there was a significant reduction of p38 signaling, as well as global transcriptional alterations consistent with the myopathic phenotype. Inhibition of Ras/MAPK pathway signaling using a MEK inhibitor rescued the HrasG12V myopathy phenotype both in vitro and in vivo, demonstrating that increased MAPK signaling is the main cause of the muscle phenotype in CS.

Download Full-text

C-terminal domain phosphorylation of ERK3 controlled by Cdk1 and Cdc14 regulates its stability in mitosis

Biochemical Journal ◽

10.1042/bj20091604 ◽

2010 ◽

Vol 428 (1) ◽

pp. 103-111 ◽

Cited By ~ 16

Author(s):

Pierre-Luc Tanguay ◽

Geneviève Rodier ◽

Sylvain Meloche

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Mitotic Cell ◽

Mitogen Activated Protein Kinase ◽

Phosphorylation Sites ◽

Dependent Manner ◽

Cell Extracts ◽

Cycle Dependent

ERK3 (extracellular-signal-regulated kinase 3) is an atypical MAPK (mitogen-activated protein kinase) that is suggested to play a role in cell-cycle progression and cellular differentiation. However, it is not known whether the function of ERK3 is regulated during the cell cycle. In the present paper, we report that ERK3 is stoichiometrically hyperphosphorylated during entry into mitosis and is dephosphorylated at the M→G1 transition. The phosphorylation of ERK3 is associated with the accumulation of the protein in mitosis. In vitro phosphorylation of a series of ERK3-deletion mutants by mitotic cell extracts revealed that phosphorylation is confined to the unique C-terminal extension of the protein. Using MS analysis, we identified four novel phosphorylation sites, Ser684, Ser688, Thr698 and Ser705, located at the extreme C-terminus of ERK3. All four sites are followed by a proline residue. We have shown that purified cyclin B-Cdk1 (cyclindependent kinase 1) phosphorylates these sites in vitro and demonstrate that Cdk1 acts as a major Thr698 kinase in vivo. Reciprocally, we found that the phosphatases Cdc14A and Cdc14B (Cdc is cell-division cycle) bind to ERK3 and reverse its C-terminal phosphorylation in mitosis. Importantly, alanine substitution of the four C-terminal phosphorylation sites markedly decreased the half-life of ERK3 in mitosis, thereby linking phosphorylation to the stabilization of the kinase. The results of the present study identify a novel regulatory mechanism of ERK3 that operates in a cell-cycle-dependent manner.

Download Full-text