Extracellular Signal-Regulated Kinase 2 Interacts with and Is Negatively Regulated by the LIM-Only Protein FHL2 in Cardiomyocytes

ABSTRACT The mitogen-activated protein kinase (MAPK) signaling pathway regulates diverse biologic functions including cell growth, differentiation, proliferation, and apoptosis. The extracellular signal-regulated kinases (ERKs) constitute one branch of the MAPK pathway that has been implicated in the regulation of cardiac differentiated growth, although the downstream mechanisms whereby ERK signaling affects this process are not well characterized. Here we performed a yeast two-hybrid screen with ERK2 bait and a cardiac cDNA library to identify novel proteins involved in regulating ERK signaling in cardiomyocytes. This screen identified the LIM-only factor FHL2 as an ERK interacting protein in both yeast and mammalian cells. In vivo, FHL2 and ERK2 colocalized in the cytoplasm at the level of the Z-line, and interestingly, FHL2 interacted more efficiently with the activated form of ERK2 than with the dephosphorylated form. ERK2 also interacted with FHL1 and FHL3 but not with the muscle LIM protein. Moreover, at least two LIM domains in FHL2 were required to mediate efficient interaction with ERK2. The interaction between ERK2 and FHL2 did not influence ERK1/2 activation, nor was FHL2 directly phosphorylated by ERK2. However, FHL2 inhibited the ability of activated ERK2 to reside within the nucleus, thus blocking ERK-dependent transcriptional responsiveness of ELK-1, GATA4, and the atrial natriuretic factor promoter. Finally, FHL2 partially antagonized the cardiac hypertrophic response induced by activated MEK-1, GATA4, and phenylephrine agonist stimulation. Collectively, these results suggest that FHL2 serves a repressor function in cardiomyocytes through its ability to inhibit ERK1/2 transcriptional coupling.

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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.

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Delivery of Sorafenib and Metformin Against Hepatocellular Carcinoma with Amphiphilic Polypeptide-based Micelles

10.21203/rs.3.rs-944726/v1 ◽

2021 ◽

Author(s):

Lanqing Cao ◽

Guangmeng Xu ◽

Hongyu He ◽

Jiannan Li

Keyword(s):

Mapk Pathway ◽

Synergistic Effects ◽

Mitogen Activated Protein Kinase ◽

High Recurrence Rate ◽

Extracellular Signal Regulated Kinase ◽

Cycle Arrest ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

Poly Ethylene

Abstract Hepatoma is a common clinical disease with poor prognosis and a high recurrence rate. Chemotherapy is important for hepatoma treatment because only a small amount of hepatoma patients are suitable for local resection, and the effects of transarterial chemoembolization (TACE) are unsatisfactory. But many limitations restrict further application of chemotherapy. In this study, sorafenib (Sor) and metformin (Met) co-loaded poly(ethylene glycol)-block-poly(L-glutamic acid-co-L-phenylalanine) (mPEG-b-P(Glu-co-Phe)) micelles were developed. Sor is a common molecular target agent which can inhibit the mitogen-activated protein kinase (MAPK) pathway to treat hepatoma clinically. Met can also regulate the MAPK pathway and inhibit the expression of the phosphorylated extracellular signal-regulated kinase (p-ERK). Moreover, both Sor and Met play important roles in cell cycle arrest. The integration of these two drugs aims to achieve synergistic effects against hepatoma. The micelles can be targeted to cancer cells and possess longer blood circulation time. The two agents can be released rapidly in the tumor sites. Both orthotopic and patient-derived xenograft (PDX) hepatoma models were developed to analyze the treatment efficacy of the Sor and Met loaded micelles. The in vivo study showed that the micelles can prevent hepatoma progression by inhibiting the expressions of p-ERK and cyclin D1. This study indicated that the Sor/Met-loaded micelles are suitable for hepatoma treatment.

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Spa2p Interacts with Cell Polarity Proteins and Signaling Components Involved in Yeast Cell Morphogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.18.7.4053 ◽

1998 ◽

Vol 18 (7) ◽

pp. 4053-4069 ◽

Cited By ~ 182

Author(s):

Yi-Jun Sheu ◽

Beatriz Santos ◽

Nathalie Fortin ◽

Christine Costigan ◽

Michael Snyder

Keyword(s):

Actin Cytoskeleton ◽

Hybrid System ◽

Mapk Pathway ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Velocity Sedimentation ◽

Mitogen Activated Protein ◽

Multiple Regions ◽

Two Hybrid

ABSTRACT The yeast protein Spa2p localizes to growth sites and is important for polarized morphogenesis during budding, mating, and pseudohyphal growth. To better understand the role of Spa2p in polarized growth, we analyzed regions of the protein important for its function and proteins that interact with Spa2p. Spa2p interacts with Pea2p and Bud6p (Aip3p) as determined by the two-hybrid system; all of these proteins exhibit similar localization patterns, and spa2Δ,pea2Δ, and bud6Δ mutants display similar phenotypes, suggesting that these three proteins are involved in the same biological processes. Coimmunoprecipitation experiments demonstrate that Spa2p and Pea2p are tightly associated with each other in vivo. Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex. Bud6p has been shown previously to interact with actin, suggesting that the 12S complex functions to regulate the actin cytoskeleton. Deletion analysis revealed that multiple regions of Spa2p are involved in its localization to growth sites. One of the regions involved in Spa2p stability and localization interacts with Pea2p; this region contains a conserved domain, SHD-II. Although a portion of Spa2p is sufficient for localization of itself and Pea2p to growth sites, only the full-length protein is capable of complementing spa2 mutant defects, suggesting that other regions are required for Spa2p function. By using the two-hybrid system, Spa2p and Bud6p were also found to interact with components of two mitogen-activated protein kinase (MAPK) pathways important for polarized cell growth. Spa2p interacts with Ste11p (MAPK kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling pathway as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK pathway; for both Mkk1p and Ste7p, the Spa2p-interacting region was mapped to the N-terminal putative regulatory domain. Bud6p interacts with Ste11p. The MEK-interacting region of Spa2p corresponds to the highly conserved SHD-I domain, which is shown to be important for mating and MAPK signaling. spa2 mutants exhibit reduced levels of pheromone signaling and an elevated level of Slt2p kinase activity. We thus propose that Spa2p, Pea2p, and Bud6p function together, perhaps as a complex, to promote polarized morphogenesis through regulation of the actin cytoskeleton and signaling pathways.

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Graded Mitogen-Activated Protein Kinase Activity Precedes Switch-Like c-Fos Induction in Mammalian Cells

Molecular and Cellular Biology ◽

10.1128/mcb.25.11.4676-4682.2005 ◽

2005 ◽

Vol 25 (11) ◽

pp. 4676-4682 ◽

Cited By ~ 71

Author(s):

Jeffrey P. MacKeigan ◽

Leon O. Murphy ◽

Christopher A. Dimitri ◽

John Blenis

Keyword(s):

Protein Kinase ◽

Cell Fate ◽

Oocyte Maturation ◽

Mammalian Cells ◽

Mapk Pathway ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Cell Fate Decisions ◽

Graded Response ◽

Mitogen Activated Protein

ABSTRACT The mitogen-activated protein kinase (MAPK) pathway is an evolutionarily conserved signaling module that controls important cell fate decisions in a variety of physiological contexts. During Xenopus oocyte maturation, the MAPK cascade converts an increasing progesterone stimulus into a switch-like, all-or-nothing response. While the importance of such switch-like behavior is widely discussed in the literature, it is not known whether the MAPK pathway in mammalian cells exhibits a switch-like or graded response. For this study, we used flow cytometry and immunofluorescence to generate single-cell measurements of MAPK signaling in Swiss 3T3 fibroblasts. In contrast to the case in Xenopus oocytes, we found that ERK activation in individual mammalian cells is not ultrasensitive and shows a graded response to changes in agonist concentration. Thus, the conserved MAPK signaling module exhibits different systems-level properties in different cellular contexts. Furthermore, the graded ERK response was converted into a more switch-like behavior at the level of immediate-early gene induction and cell cycle progression. Thus, while MAPK signaling is involved in all-or-nothing cell fate decisions for both Xenopus oocyte maturation and mammalian fibroblast proliferation, the underlying mechanisms responsible for the switch-like nature of the cellular responses are different in these two systems, with the mechanism appearing to lie downstream of the kinase cascade in mammalian fibroblasts.

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Exploiting codon usage identifies RpS21 as an in vivo signal strength-dependent Ras/MAPK regulator

10.1101/650630 ◽

2019 ◽

Author(s):

Jessica K. Sawyer ◽

Zahra Kabiri ◽

Ruth A. Montague ◽

Sarah V. Paramore ◽

Erez Cohen ◽

...

Keyword(s):

Codon Usage ◽

Mapk Pathway ◽

Mapk Signaling ◽

Ribosomal Gene ◽

Mitogen Activated Protein Kinase ◽

Biological Processes ◽

Cell Tissue ◽

Mitogen Activated Protein ◽

Multiple Cell

ABSTRACTSignal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. Here, by altering codon usage as a novel platform to control signaling output, we screened the Drosophila genome for modifiers specific to either weak or strong Ras-driven eye phenotypes. We mapped the underlying gene from one modifier to the ribosomal gene RpS21. RpS21 preferentially influences weak Ras/MAPK signaling outputs, and negatively regulates Ras/MAPK in multiple cell/tissue and signaling settings. In turn, MAPK signaling may regulate its own negative feedback by promoting RpS21 expression. These data show that codon usage manipulation can identify output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator.

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Mitogen-activated protein kinase (MAPK) blockade of bovine preimplantation embryogenesis requires inhibition of both p38 and extracellular signal-regulated kinase (ERK) pathways

Reproduction ◽

10.1530/rep.1.00554 ◽

2005 ◽

Vol 130 (1) ◽

pp. 41-51 ◽

Cited By ~ 22

Author(s):

Pavneesh Madan ◽

Michele D Calder ◽

Andrew J Watson

Keyword(s):

P38 Mapk ◽

Mapk Signaling ◽

Blastocyst Stage ◽

Mitogen Activated Protein Kinase ◽

Erk Signaling ◽

Bovine Embryos ◽

Blastocyst Formation ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal ◽

Mitogen Activated Protein

Blastocyst formation, as a critical period during development, is an effective indicator of embryonic health and reproductive efficiency. Out of a number of mechanisms underlying blastocyst formation, highly conserved mitogen-activated protein kinase (MAPK) signaling has emerged as a major mechanism involved in regulating murine preimplantation embryo development. The objective of our study was to ascertain the role of MAPK signaling in regulating bovine development to the blastocyst stage. Using reverse transcriptase PCR and immunohistochemical staining procedures we have demonstrated that mRNA transcripts and polypeptides encoding p38 MAPK pathway constituents are detectable in preimplantation bovine embryos from the one-cell to the blastocyst stage. Further, the effects on bovine embryo development following inhibition of p38 α/β and extracellular signal-regulated kinase (ERK) signaling by treatment with SB220025 and U0126, respectively, were investigated. Eight-cell bovine embryos (50 per group; three replicates) were placed into treatments consisting of synthetic oviductal fluid (SOF) medium: SOF + SB202474 (inactive analogue), SOF + SB220025, SOF + U0124 (inactive analogue), SOF + U0126, and SOF + SB220025 + U0126. Inhibition of p38 MAPK or ERK signaling individually did not affect development to the blastocyst stage. However, when both pathways were blocked simultaneously there was a significant reduction (P< 0.05) in blastocyst formation, cell number and immunofluorescence of phosphorylated downstream pathway constituents. We have determined that, in variance to what was observed during murine preimplantation development, bovine early embryos progress at normal frequencies to the blastocyst stage in the presence of p38 MAPK inhibitors.

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Combined VEGFR and MAPK pathway inhibition in angiosarcoma

Scientific Reports ◽

10.1038/s41598-021-88703-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Michael J. Wagner ◽

Yasmin A. Lyons ◽

Jean H. Siedel ◽

Robert Dood ◽

Archana S. Nagaraja ◽

...

Keyword(s):

Protein Kinase ◽

Mapk Pathway ◽

Combined Treatment ◽

Mek Inhibitor ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

In Vivo Model ◽

Mitogen Activated Protein

AbstractAngiosarcoma is an aggressive malignancy of endothelial cells that carries a high mortality rate. Cytotoxic chemotherapy can elicit clinical responses, but the duration of response is limited. Sequencing reveals multiple mutations in angiogenesis pathways in angiosarcomas, particularly in vascular endothelial growth factor (VEGFR) and mitogen-activated protein kinase (MAPK) signaling. We aimed to determine the biological relevance of these pathways in angiosarcoma. Tissue microarray consisting of clinical formalin-fixed paraffin embedded tissue archival samples were stained for phospho- extracellular signal-regulated kinase (p-ERK) with immunohistochemistry. Angiosarcoma cell lines were treated with the mitogen-activated protein kinase kinase (MEK) inhibitor trametinib, pan-VEGFR inhibitor cediranib, or combined trametinib and cediranib and viability was assessed. Reverse phase protein array (RPPA) was performed to assess multiple oncogenic protein pathways. SVR angiosarcoma cells were grown in vivo and gene expression effects of treatment were assessed with whole exome RNA sequencing. MAPK signaling was found active in over half of clinical angiosarcoma samples. Inhibition of MAPK signaling with the MEK inhibitor trametinib decreased the viability of angiosarcoma cells. Combined inhibition of the VEGF and MAPK pathways with cediranib and trametinib had an additive effect in in vitro models, and a combinatorial effect in an in vivo model. Combined treatment led to smaller tumors than treatment with either agent alone. RNA-seq demonstrated distinct expression signatures between the trametinib treated tumors and those treated with both trametinib and cediranib. These results indicate a clinical study of combined VEGFR and MEK inhibition in angiosarcoma is warranted.

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Exploiting codon usage identifies intensity-specific modifiers of Ras/MAPK signaling in vivo

PLoS Genetics ◽

10.1371/journal.pgen.1009228 ◽

2020 ◽

Vol 16 (12) ◽

pp. e1009228

Author(s):

Jessica K. Sawyer ◽

Zahra Kabiri ◽

Ruth A. Montague ◽

Scott R. Allen ◽

Rebeccah Stewart ◽

...

Keyword(s):

Codon Usage ◽

Mapk Pathway ◽

Mapk Signaling ◽

Ribosomal Gene ◽

Mitogen Activated Protein Kinase ◽

Signal Transduction Pathways ◽

Biological Processes ◽

Multiple Contexts ◽

Mitogen Activated Protein

Signal transduction pathways are intricately fine-tuned to accomplish diverse biological processes. An example is the conserved Ras/mitogen-activated-protein-kinase (MAPK) pathway, which exhibits context-dependent signaling output dynamics and regulation. Here, by altering codon usage as a novel platform to control signaling output, we screened the Drosophila genome for modifiers specific to either weak or strong Ras-driven eye phenotypes. Our screen enriched for regions of the genome not previously connected with Ras phenotypic modification. We mapped the underlying gene from one modifier to the ribosomal gene RpS21. In multiple contexts, we show that RpS21 preferentially influences weak Ras/MAPK signaling outputs. These data show that codon usage manipulation can identify new, output-specific signaling regulators, and identify RpS21 as an in vivo Ras/MAPK phenotypic regulator.

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TORC1 Suppression Predicts Responsiveness to RAF and MEK Inhibition in BRAF-Mutant Melanoma

Science Translational Medicine ◽

10.1126/scitranslmed.3005753 ◽

2013 ◽

Vol 5 (196) ◽

pp. 196ra98-196ra98 ◽

Cited By ~ 86

Author(s):

Ryan B. Corcoran ◽

Stephen Michael Rothenberg ◽

Aaron N. Hata ◽

Anthony C. Faber ◽

Adriano Piris ◽

...

Keyword(s):

Protein Kinase ◽

Kinase Inhibitors ◽

Progression Free Survival ◽

Needle Aspiration ◽

Mapk Signaling ◽

Mitogen Activated Protein Kinase ◽

Mek Inhibitors ◽

Extracellular Signal ◽

Mitogen Activated Protein

RAF and MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase) inhibitors are effective in treating patients with BRAF-mutant melanoma. However, most responses are partial and short-lived, and many patients fail to respond at all. We found that suppression of TORC1 activity in response to RAF or MEK inhibitors, as measured by decreased phosphorylation of ribosomal protein S6 (P-S6), effectively predicted induction of cell death by the inhibitor in BRAF-mutant melanoma cell lines. In resistant melanomas, TORC1 activity was maintained after treatment with RAF or MEK inhibitors, in some cases despite robust suppression of mitogen-activated protein kinase (MAPK) signaling. In in vivo mouse models, suppression of TORC1 after MAPK inhibition was necessary for induction of apoptosis and tumor response. Finally, in paired biopsies obtained from patients with BRAF-mutant melanoma before treatment and after initiation of RAF inhibitor therapy, P-S6 suppression predicted significantly improved progression-free survival. Such a change in P-S6 could be readily monitored in real time by serial fine-needle aspiration biopsies, making quantitation of P-S6 a valuable biomarker to guide treatment in BRAF-mutant melanoma.

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Bromamine T (BAT) Exerts Stronger Anti-Cancer Properties than Taurine (Tau)

Cancers ◽

10.3390/cancers13020182 ◽

2021 ◽

Vol 13 (2) ◽

pp. 182

Author(s):

Stella Baliou ◽

Maria Goulielmaki ◽

Petros Ioannou ◽

Christina Cheimonidi ◽

Ioannis P. Trougakos ◽

...

Keyword(s):

Cytotoxic Effect ◽

Human Colon ◽

Mitogen Activated Protein Kinase ◽

Mitochondrial Apoptosis ◽

Therapeutic Modalities ◽

Cancer Pathogenesis ◽

Extracellular Signal ◽

Anti Cancer ◽

Mitogen Activated Protein

Background: Taurine (Tau) ameliorates cancer pathogenesis. Researchers have focused on the functional properties of bromamine T (BAT), a stable active bromine molecule. Both N-bromotaurine (TauNHBr) and BAT exert potent anti-inflammatory properties, but the landscape remains obscure concerning the anti-cancer effect of BAT. Methods: We used Crystal Violet, colony formation, flow cytometry and Western blot experiments to evaluate the effect of BAT and Tau on the apoptosis and autophagy of cancer cells. Xenograft experiments were used to determine the in vivo cytotoxicity of either agent. Results: We demonstrated that both BAT and Tau inhibited the growth of human colon, breast, cervical and skin cancer cell lines. Among them, BAT exerted the greatest cytotoxic effect on both RKO and MDA-MB-468 cells. In particular, BAT increased the phosphorylation of c-Jun N-terminal kinases (JNK½), p38 mitogen-activated protein kinase (MAPK), and extracellular-signal-regulated kinases (ERK½), thereby inducing mitochondrial apoptosis and autophagy in RKO cells. In contrast, Tau exerted its cytotoxic effect by upregulating JNK½ forms, thus triggering mitochondrial apoptosis in RKO cells. Accordingly, colon cancer growth was impaired in vivo. Conclusions: BAT and Tau exerted their anti-tumor properties through the induction of (i) mitochondrial apoptosis, (ii) the MAPK family, and iii) autophagy, providing novel anti-cancer therapeutic modalities.

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