scholarly journals A Raf-1 Mutant That Dissociates MEK/Extracellular Signal-Regulated Kinase Activation from Malignant Transformation and Differentiation but Not Proliferation

2003 ◽  
Vol 23 (6) ◽  
pp. 1983-1993 ◽  
Author(s):  
Amardeep S. Dhillon ◽  
Sharon Meikle ◽  
Carole Peyssonnaux ◽  
Joan Grindlay ◽  
Christian Kaiser ◽  
...  
Keyword(s):  
Cell Transformation ◽  
Primary Cultures ◽  
Erk Pathway ◽  
Morphological Alterations ◽  
Extracellular Signal Regulated Kinase ◽  
Amino Terminal ◽  
3T3 Fibroblasts ◽  
Extracellular Signal ◽  
Accelerated Proliferation ◽  
Nih 3T3

ABSTRACT It is widely thought that the biological outcomes of Raf-1 activation are solely attributable to the activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. However, an increasing number of reports suggest that some Raf-1 functions are independent of this pathway. In this report we show that mutation of the amino-terminal 14-3-3 binding site of Raf-1 uncouples its ability to activate the MEK/ERK pathway from the induction of cell transformation and differentiation. In NIH 3T3 fibroblasts and COS-1 cells, mutation of serine 259 resulted in Raf-1 proteins which activated the MEK/ERK pathway as efficiently as v-Raf. However, in contrast to v-Raf, RafS259 mutants failed to transform. They induced morphological alterations and slightly accelerated proliferation in NIH 3T3 fibroblasts but were not tumorigenic in mice and behaved like wild-type Raf-1 in transformation assays measuring loss of contact inhibition or anchorage-independent growth. Curiously, the RafS259 mutants inhibited focus induction by an activated MEK allele, suggesting that they can hyperactivate negative-feedback pathways. In primary cultures of postmitotic chicken neuroretina cells, RafS259A was able to sustain proliferation to a level comparable to that sustained by the membrane-targeted transforming Raf-1 protein, RafCAAX. In contrast, RafS259A was only a poor inducer of neurite formation in PC12 cells in comparison to RafCAAX. Thus, RafS259 mutants genetically separate MEK/ERK activation from the ability of Raf-1 to induce transformation and differentiation. The results further suggest that RafS259 mutants inhibit signaling pathways required to promote these biological processes.

scholarly journals Analgesic Efficacy of a Combination of Fentanyl and a Japanese Herbal Medicine “Yokukansan” in Rats with Acute Inflammatory Pain

Medicines ◽  
2020 ◽  
Vol 7 (12) ◽  
pp. 75
Author(s):  
Yuko Akanuma ◽  
Mami Kato ◽  
Yasunori Takayama ◽  
Hideshi Ikemoto ◽  
Naoki Adachi ◽  
...  
Keyword(s):  
Inflammatory Pain ◽  
Late Phase ◽  
Analgesic Efficacy ◽  
Opioid Tolerance ◽  
High Dose ◽  
Erk Pathway ◽  
Single High Dose ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Combined Use

Background: Fentanyl can induce acute opioid tolerance and postoperative hyperalgesia when administered at a single high dose; thus, this study examined the analgesic efficacy of a combination of fentanyl and Yokukansan (YKS). Methods: Rats were divided into control, formalin-injected (FOR), YKS-treated+FOR (YKS), fentanyl-treated+FOR (FEN), and YKS+FEN+FOR (YKS+FEN) groups. Acute pain was induced via subcutaneous injection of formalin into the paw. The time engaged in pain-related behavior was measured. Results: In the early (0–10 min) and intermediate (10–20 min) phases, pain-related behavior in the YKS+FEN group was significantly inhibited compared with the FOR group. In the late phase (20–60 min), pain-related behavior in the FEN group was the longest and significantly increased compared with the YKS group. We explored the influence on the extracellular signal-regulated kinase (ERK) pathway in the spinal cord, and YKS suppressed the phosphorylated ERK expression, which may be related to the analgesic effect of YKS in the late phase. Conclusions: These findings suggest that YKS could reduce the use of fentanyl and combined use of YKS and fentanyl is considered clinically useful.

scholarly journals Calmodulin-Independent Coordination of Ras and Extracellular Signal-Regulated Kinase Activation by Ras-GRF2

2000 ◽  
Vol 20 (8) ◽  
pp. 2727-2733 ◽  
Author(s):  
Carmen L. de Hoog ◽  
Wing-Tze Fan ◽  
Marni D. Goldstein ◽  
Michael F. Moran ◽  
C. Anne Koch
Keyword(s):  
Map Kinase ◽  
Mutational Analysis ◽  
Ras Signaling ◽  
Sequence Motifs ◽  
Nucleotide Exchange ◽  
Ph Domain ◽  
Extracellular Signal Regulated Kinase ◽  
Amino Terminal ◽  
Extracellular Signal

ABSTRACT Ras-GRF2 (GRF2) is a widely expressed, calcium-activated regulator of the small-type GTPases Ras and Rac. It is a multidomain protein composed of several recognizable sequence motifs in the following order (NH2 to COOH): pleckstrin homology (PH), coiled-coil, ilimaquinone (IQ), Dbl homology (DH), PH, REM (Ras exchanger motif), PEST/destruction box, Cdc25. The DH and Cdc25 domains possess guanine nucleotide exchange factor (GEF) activity and interact with Rac and Ras, respectively. The REM-Cdc25 region was found to be sufficient for maximal activation of Ras in vitro and in vivo caused Ras and extracellular signal-regulated kinase (ERK) activation independent of calcium signals, suggesting that, at least when expressed ectopically, it contains all of the determinants required to access and activate Ras signaling. Additional mutational analysis of GRF2 indicated that the carboxyl PH domain imparts a modest inhibitory effect on Ras GEF activity and probably normally participates in intermolecular interactions. A variant of GRF2 missing the Cdc25 domain did not activate Ras and functions as an inhibitor of wild-type GRF2, presumably by competing for interactions with molecules other than calmodulin, Ras, and ligands of the PH domain. The binding of calmodulin was found to require several amino-terminal domains of GRF2 in addition to the IQ sequence, and no correlation between calmodulin binding by GRF2 and its ability to directly activate Ras and indirectly stimulate the mitogen-activated protein (MAP) kinase ERK in response to calcium was found. The precise role of the GRF2-calmodulin association, therefore, remains to be determined. A GRF2 mutant missing the IQ sequence was competent for Ras activation but failed to couple this to stimulation of the ERK pathway. This demonstrates that Ras-GTP formation is not sufficient for MAP kinase signaling. We conclude that in addition to directly activating Ras, GRF2, and likely other GEFs, promote the assembly of a protein network able to couple the GTPase with particular effectors.

scholarly journals RACK1 Targets the Extracellular Signal-Regulated Kinase/Mitogen-Activated Protein Kinase Pathway To Link Integrin Engagement with Focal Adhesion Disassembly and Cell Motility

2007 ◽  
Vol 27 (23) ◽  
pp. 8296-8305 ◽  
Author(s):  
Tomas Vomastek ◽  
Marcin P. Iwanicki ◽  
Hans-Joerg Schaeffer ◽  
Adel Tarcsafalvi ◽  
J. Thomas Parsons ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Mitogen Activated Protein Kinase ◽  
Erk Pathway ◽  
Large Measure ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Erk Activity

ABSTRACT The extracellular signal-regulated kinase (ERK) cascade is activated in response to a multitude of extracellular signals and converts these signals into a variety of specific biological responses, including cell differentiation, cell movement, cell division, and apoptosis. The specificity of the biological response is likely to be controlled in large measure by the localization of signaling, thus enabling ERK activity to be directed towards specific targets. Here we show that the RACK1 scaffold protein functions specifically in integrin-mediated activation of the mitogen-activated protein kinase/ERK cascade and targets active ERK to focal adhesions. We found that RACK1 associated with the core kinases of the ERK pathway, Raf, MEK, and ERK, and that attenuation of RACK1 expression resulted in a decrease in ERK activity in response to adhesion but not in response to growth factors. RACK1 silencing also caused a reduction of active ERK in focal adhesions, an increase in focal adhesion length, a decreased rate of focal adhesion disassembly, and decreased motility. Our data further suggest that focal adhesion kinase is an upstream activator of the RACK1/ERK pathway. We suggest that RACK1 tethers the ERK pathway core kinases and channels signals from upstream activation by integrins to downstream targets at focal adhesions.

scholarly journals Distinct Cell Cycle Timing Requirements for Extracellular Signal-Regulated Kinase and Phosphoinositide 3-Kinase Signaling Pathways in Somatic Cell Mitosis

2002 ◽  
Vol 22 (20) ◽  
pp. 7226-7241 ◽  
Author(s):  
Elisabeth C. Roberts ◽  
Paul S. Shapiro ◽  
Theresa Stines Nahreini ◽  
Gilles Pages ◽  
Jacques Pouyssegur ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cyclin B1 ◽  
Erk Pathway ◽  
Cycle Progression ◽  
Mitotic Entry ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Phosphoinositide 3 Kinase ◽  
And Function

ABSTRACT Mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase (PI3K) pathways are necessary for cell cycle progression into S phase; however the importance of these pathways after the restriction point is poorly understood. In this study, we examined the regulation and function of extracellular signal-regulated kinase (ERK) and PI3K during G2/M in synchronized HeLa and NIH 3T3 cells. Phosphorylation and activation of both the MAP kinase kinase/ERK and PI3K/Akt pathways occur in late S and persist until the end of mitosis. Signaling was rapidly reversed by cell-permeable inhibitors, indicating that both pathways are continuously activated and rapidly cycle between active and inactive states during G2/M. The serum-dependent behavior of PI3K/Akt versus ERK pathway activation indicates that their mechanisms of regulation differ during G2/M. Effects of cell-permeable inhibitors and dominant-negative mutants show that both pathways are needed for mitotic progression. However, inhibiting the PI3K pathway interferes with cdc2 activation, cyclin B1 expression, and mitotic entry, whereas inhibiting the ERK pathway interferes with mitotic entry but has little effect on cdc2 activation and cyclin B1 and retards progression from metaphase to anaphase. Thus, our study provides novel evidence that ERK and PI3K pathways both promote cell cycle progression during G2/M but have different regulatory mechanisms and function at distinct times.

scholarly journals Epstein-Barr Virus Latent Membrane Protein 2A Regulates c-Jun Protein through Extracellular Signal-Regulated Kinase

2002 ◽  
Vol 76 (18) ◽  
pp. 9556-9561 ◽  
Author(s):  
Shao-Yin Chen ◽  
Jean Lu ◽  
Yin-Chu Shih ◽  
Ching-Hwa Tsai
Keyword(s):  
Protein Kinase ◽  
Epstein Barr Virus ◽  
Latent Membrane Protein ◽  
Erk Pathway ◽  
Latent Membrane Protein 2A ◽  
Extracellular Signal Regulated Kinase ◽  
Barr Virus ◽  
Extracellular Signal ◽  
Infected Cells ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is widely expressed in both EBV-infected cells and EBV-associated malignancies. However, the function of LMP2A is still veiled. In this study, LMP2A was found to induce the kinase activities of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase/stress-activated protein kinase JNK/SAPK. Furthermore, the downstream effector c-Jun showed hyperphosphorylation under LMP2A expression. The phosphorylation could be inhibited by the ERK pathway inhibitor PD98059, indicating that ERK may contribute to the phosphorylation of c-Jun in LMP2A-expressing cells. The impact on c-Jun phosphorylation by mitogen-activated protein kinase (MAPK) is suggested to increase c-Jun protein stability, and this was also observed in LMP2A-expressing cells by a protein synthesis inhibition assay. Moreover, LMP2A-induced cell invasion was inhibited in the presence of the ERK pathway inhibitor. Taken together, we suggest that LMP2A may exploit MAPK kinases and affect both the phosphorylation and stability of c-Jun protein. Additionally, LMP2A may thereby promote the mobility of the cells. In doing so, it may enhance the mobility of EBV-infected cells and contribute to the metastatic process of malignant cells. Here we demonstrated the first evidence of LMP2A-induced migration and the underlying pathways accounting for it.

scholarly journals The Molecular Chaperone Heat Shock Protein 70 Controls Liver Cancer Initiation and Progression by Regulating Adaptive DNA Damage and Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Signaling Pathways

2019 ◽  
Vol 39 (9) ◽  
Author(s):  
Wonkyoung Cho ◽  
Xiongjie Jin ◽  
Junfeng Pang ◽  
Yan Wang ◽  
Nahid F. Mivechi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Heat Shock ◽  
Protein Kinase ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Cell Transformation ◽  
Mitogen Activated Protein Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Delineating the mechanisms that drive hepatic injury and hepatocellular carcinoma (HCC) progression is critical for development of novel treatments for recurrent and advanced HCC but also for the development of diagnostic and preventive strategies. Heat shock protein 70 (HSP70) acts in concert with several cochaperones and nucleotide exchange factors and plays an essential role in protein quality control that increases survival by protecting cells against environmental stressors. Specifically, the HSP70-mediated response has been implicated in the pathogenesis of cancer, but the specific mechanisms by which HSP70 may support malignant cell transformation remains to be fully elucidated. Here, we show that genetic ablation of HSP70 markedly impairs HCC initiation and progression by distinct but overlapping pathways. This includes the potentiation of the carcinogen-induced DNA damage response, at the tumor initiation stage, to increase the p53-dependent surveillance response leading to the cell cycle exit or death of genomically damaged differentiated pericentral hepatocytes, and this may also prevent their conversion into more proliferating HCC progenitor cells. Subsequently, activation of a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) negative feedback pathway diminishes oncogenic signals, thereby attenuating premalignant cell transformation and tumor progression. Modulation of HSP70 function may be a strategy for interfering with oncogenic signals driving liver cell transformation and tumor progression, thus providing an opportunity for human cancer control.

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