Multiple Mitogen-activated Protein Kinase Research Articles

The Function of ASK1 in Sepsis and Stress-Induced Disorders.

Apoptosis signal-regulating kinase 1 (ASK1) is a serine-threonine kinase that is ubiquitously expressed in nucleated cells and is responsible for the activation of multiple mitogen-activated protein kinases (MAPK) to regulate cell stress. Activation of ASK1 via cellular stress leads to activation of downstream signaling components, activation of transcription factors, and proinflammatory cytokine production. ASK1 is also expressed in anucleate platelets and is a key player in platelet activation as it is important for signaling. Interestingly, the mechanism of ASK1 activation is cell type-dependent. In this review we will explore how ASK1 regulates a variety of cellular processes from innate immune function to thrombosis and hemostasis. We will discuss how ASK1 influences FcγRIIA-mediated platelet reactivity and how that reactivity drives platelet clearance. Furthermore, we will explore the role of ASK1 in thromboxane (TxA2) generation, which highlights differences in the way ASK1 functions in mouse and human platelets.

Multiple cancers escape from multiple MAPK pathway inhibitors and use DNA replication stress signaling to tolerate aberrant cell cycles.

Many cancers harbor pro-proliferative mutations of the mitogen-activated protein kinase (MAPK) pathway. In BRAF-driven melanoma cells treated with BRAF inhibitors, subpopulations of cells escape drug-induced quiescence through a nongenetic manner of adaptation and resume slow proliferation. Here, we found that this phenomenon is common to many cancer types driven by EGFR, KRAS, or BRAF mutations in response to multiple, clinically approved MAPK pathway inhibitors. In 2D cultures and 3D spheroid models of various cancer cell lines, a subset of cells escaped drug-induced quiescence within 4 days to resume proliferation. These "escapee" cells exhibited DNA replication deficits, accumulated DNA lesions, and mounted a stress response that depended on the ataxia telangiectasia and RAD3-related (ATR) kinase. We further identified that components of the Fanconi anemia (FA) DNA repair pathway are recruited to sites of mitotic DNA synthesis (MiDAS) in escapee cells, enabling successful completion of cell division. Analysis of patient tumor samples and clinical data correlated disease progression with an increase in DNA replication stress response factors. Our findings suggest that many MAPK pathway-mutant cancers rapidly escape drug action and that suppressing early stress tolerance pathways may achieve more durable clinical responses to MAPK pathway inhibitors.

Phosphorylation of mixed lineage kinase MLK3 by cyclin-dependent kinases CDK1 and CDK2 controls ovarian cancer cell division

Mixed lineage kinase 3 (MLK3) is a serine/threonine mitogen-activated protein kinase kinase kinase that promotes the activation of multiple mitogen-activated protein kinase pathways and is required for invasion and proliferation of ovarian cancer cells. Inhibition of MLK activity causes G2/M arrest in HeLa cells; however, the regulation of MLK3 during ovarian cancer cell cycle progression is not known. Here, we found that MLK3 is phosphorylated in mitosis and that inhibition of cyclin-dependent kinase 1 (CDK1) prevented MLK3 phosphorylation. In addition, we observed that c-Jun N-terminal kinase, a downstream target of MLK3 and a direct target of MKK4 (SEK1), was activated in G2 phase when CDK2 activity is increased and then inactivated at the beginning of mitosis concurrent with the increase in CDK1 and MLK3 phosphorylation. Using in vitro kinase assays and phosphomutants, we determined that CDK1 phosphorylates MLK3 on Ser548 and decreases MLK3 activity during mitosis, whereas CDK2 phosphorylates MLK3 on Ser770 and increases MLK3 activity during G1/S and G2 phases. We also found that MLK3 inhibition causes a reduction in cell proliferation and a cell cycle arrest in ovarian cancer cells, suggesting that MLK3 is required for ovarian cancer cell cycle progression. Taken together, our results suggest that phosphorylation of MLK3 by CDK1 and CDK2 is important for the regulation of MLK3 and c-Jun N-terminal kinase activities during G1/S, G2, and M phases in ovarian cancer cell division.

Time-resolved phosphoproteome and proteome analysis reveals kinase signaling on master transcription factors during myogenesis

SummaryMyogenesis is governed by signaling networks that are tightly regulated in a time-dependent manner. Although different protein kinases have been identified, knowledge of the global signaling networks and their downstream substrates during myogenesis remains incomplete. Here, we map the myogenic differentiation of C2C12 cells using phosphoproteomics and proteomics. From these data, we infer global kinase activity and predict the substrates that are involved in myogenesis. We found that multiple mitogen-activated protein kinases (MAPKs) mark the initial wave of signaling cascades. Further phosphoproteomic and proteomic profiling with MAPK1/3 and MAPK8/9 specific inhibitions unveil their shared and distinctive roles in myogenesis. Lastly, we identified and validated the transcription factor nuclear factor 1 X-type (NFIX) as a novel MAPK1/3 substrate and demonstrated the functional impact of NFIX phosphorylation on myogenesis. Altogether, these data characterize the dynamics, interactions, and downstream control of kinase signaling networks during myogenesis on a global scale.

Multiple phosphorylation events of the mitochondrial membrane protein TTM1 regulate cell death during senescence.

The role of mitochondria in programmed cell death (PCD) during animal growth and development is well documented, but much less is known for plants. We previously showed that the Arabidopsis thaliana triphosphate tunnel metalloenzyme (TTM) proteins TTM1 and TTM2 are tail-anchored proteins that localize in the mitochondrial outer membrane and participate in PCD during senescence and immunity, respectively. Here, we show that TTM1 is specifically involved in senescence induced by abscisic acid (ABA). Moreover, phosphorylation of TTM1 by multiple mitogen-activated protein (MAP) kinases regulates its function and turnover. A combination of proteomics and in vitro kinase assays revealed three major phosphorylation sites of TTM1 (Ser10, Ser437, and Ser490). Ser437, which is phosphorylated upon perception of senescence cues such as ABA and prolonged darkness, is phosphorylated by the MAP kinases MPK3 and MPK4, and Ser437 phosphorylation is essential for TTM1 function in senescence. These MPKs, together with three additional MAP kinases (MPK1, MPK7, and MPK6), also phosphorylate Ser10 and Ser490, marking TTM1 for protein turnover, which likely prevents uncontrolled cell death. Taken together, our results show that multiple MPKs regulate the function and turnover of the mitochondrial protein TTM1 during senescence-associated cell death, revealing a novel link between mitochondria and PCD.

Nobiletin Inhibits Inflammatory Reaction in Interleukin-1β-Stimulated Human Periodontal Ligament Cells.

The immune response in periodontal lesions is involved in the progression of periodontal disease. Therefore, it is important to find a bioactive substance that has anti-inflammatory effects in periodontal lesions. This study aimed to examine if nobiletin, which is found in the peel of citrus fruits, could inhibit inflammatory responses in interleukin (IL)-1β-stimulated human periodontal ligament cells (HPDLCs). The release of cytokines (IL-6, IL-8, CXCL10, CCL20, and CCL2) and matrix metalloproteinases (MMP-1 and MMP-3) was assessed by ELISA. The expression of cell adhesion molecules (ICAM-1and VCAM-1) and the activation of signal transduction pathways (nuclear factor (NF)-κB, mitogen-activated protein kinases (MAPKs) and protein kinase B (Akt)) in HPDLCs were detected by Western blot analysis. Our experiments revealed that nobiletin decreased the expression of inflammatory cytokines, cell adhesion molecules, and MMPs in IL-1β-stimulated HPDLCs. Moreover, we revealed that nobiletin treatment could suppress the activation of the NF-κB, MAPKs, and Akt pathways. These findings indicate that nobiletin could inhibit inflammatory reactions in IL-1β-stimulated HPDLCs by inhibiting multiple signal transduction pathways, including NF-κB, MAPKs, and Akt.

Integrated molecular characterisation of the MAPK pathways in human cancers reveals pharmacologically vulnerable mutations and gene dependencies

The mitogen-activated protein kinase (MAPK) pathways are crucial regulators of the cellular processes that fuel the malignant transformation of normal cells. The molecular aberrations which lead to cancer involve mutations in, and transcription variations of, various MAPK pathway genes. Here, we examine the genome sequences of 40,848 patient-derived tumours representing 101 distinct human cancers to identify cancer-associated mutations in MAPK signalling pathway genes. We show that patients with tumours that have mutations within genes of the ERK-1/2 pathway, the p38 pathways, or multiple MAPK pathway modules, tend to have worse disease outcomes than patients with tumours that have no mutations within the MAPK pathways genes. Furthermore, by integrating information extracted from various large-scale molecular datasets, we expose the relationship between the fitness of cancer cells after CRISPR mediated gene knockout of MAPK pathway genes, and their dose-responses to MAPK pathway inhibitors. Besides providing new insights into MAPK pathways, we unearth vulnerabilities in specific pathway genes that are reflected in the re sponses of cancer cells to MAPK targeting drugs: a revelation with great potential for guiding the development of innovative therapies.

Anti-neuroinflammatory effects of Ginkgo biloba extract EGb761 in LPS-activated primary microglial cells

BackgroundNeuroinflammation is a key factor of Alzheimer's disease (AD) and other neurodegenerative conditions. Microglia are the resident mononuclear immune cells of the central nervous system (CNS). They play an essential role in the maintenance of homeostasis and responses to neuroinflammation. Ginkgo biloba extract EGb 761 is one of the most commonly used natural medicines owing to its established efficacy and remarkable biological activities especially in respect to CNS diseases. However, only few studies have addressed the effects and mechanisms of Ginkgo biloba extract in microglia activation. MethodsWe measured the production of pro-inflammatory mediators and cytokines by ELISA and analyzed gene expressions by qRT-PCR and Western Blot in LPS treated cultured primary rat microglia. ResultsThe Ginkgo biloba extract EGb 761 significantly inhibited the release of prostaglandin E2 (PGE2) and differentially regulated the levels of pro-inflammatory cytokines. The inhibition of LPS-induced PGE2 release in primary microglia was partially dependent on reduced protein synthesis of mPGES-1 and the reduction in the activation of cytosolic phospholipase A2 (cPLA2) without altering COX-2 enzymatic activity, inhibitor of kappa B alpha (IkappaBalpha) degradation, and the activation of multiple mitogen activated protein kinases (MAPKs). Altogether, we showed that EGb 761 reduces neuro-inflammatory activation in primary microglial cells by targeting PGE2 release and cytokines. ConclusionGinkgo biloba extract EGb 761 displayed anti-neuroinflammatory activity in LPS-activated primary microglia cells. EGb 761 was able to reduce neuroinflammatory activation by targeting the COX/PGE2 pathway. This effect might contribute to the established clinical cognitive efficacy in Alzheimer's disease, vascular and mixed dementia.

Pretreatment of ferulic acid attenuates inflammation and oxidative stress in a rat model of lipopolysaccharide-induced acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a fatal clinical condition that can be caused by pulmonary and non-pulmonary diseases. Oxidative stress and inflammation play key roles in the development of ARDS. In this study, we investigated whether ferulic acid (FA), an anti-oxidant, was beneficial for prophylaxis of ARDS. We established an ARDS rat model using lipopolysaccharide (LPS) administration. Lung injury was assessed by lung wet/dry ratio and broncho-alveolar lavage fluid (BALF) analysis. Hematoxylin and eosin staining was performed to evaluate the histological changes of the lungs. Enzyme-linked immunosorbent assay (ELISA) and immunoblotting were performed to detect proteins in BALF and lung tissue, respectively. Pulmonary function was determined by testing the oxygen level in BALF. FA pretreatment significantly alleviated LPS-induced pulmonary histological changes. FA reversed LPS-induced changes of lung wet/dry ratio, total protein in BALF, P(A-a)O2, and PaO2/FiO2. In addition, LPS dramatically up-regulated the secretion of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-10 in BALF (P < 0.01). However, pretreatment of FA significantly improved LPS-induced inflammation. We found that FA indeed reduced oxidative stress in the lungs by testing malondialdehyde level, myeloperoxidase level, and total anti-oxidant capacity. We also proved that FA inactivated multiple mitogen-activated protein kinase signaling pathways in the lungs. In conclusion, FA alleviated LPS-induced ARDS through its anti-inflammatory and anti-oxidant activities.

Silymarin mitigates lung impairments in a rat model of acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a deadly disease and lacks effective treatments. Inflammation and oxidative stress play key roles in ARDS development. We aimed to evaluate the efficacy of pretreatment of silymarin, which has capacities of anti-inflammatory and anti-oxidative stress, in ARDS. We used lipopolysaccharide (LPS) to generate an ARDS rat model, which was pretreated with silymarin. Lung wet/dry ratio and broncho-alveolar lavage fluid (BALF) analyses were performed. Histological changes of the lungs were evaluated using hematoxylin and eosin staining. Cells and proteins in BALF were determined. Protein levels in the lungs were assessed using immunoblotting. LPS administration significantly caused an increased lung wet/dry ratio, an elevated protein level in BALF, and an impaired pulmonary function in the rats. Silymarin mitigated these changes in a dose-dependent manner. Silymarin ameliorated LPS-induced histological changes of the lungs, and reduced infiltration of lymphocytes, macrophages, and neutrophils. Consistently, concentrations of pro-inflammatory cytokines such as interferon-γ, interleukin (IL)-6, and tumor necrosis factor (TNF)-α were increased, while that of anti-inflammatory cytokine IL-10 was decreased in BALF. Additionally, silymarin pretreatment partially inactivated multiple mitogen-activated protein kinase signaling pathways in the lungs. Silymarin mitigated LPS-induced lung impairments by down-regulating inflammation in a rat model.

S1PR1 mediates anti‑apoptotic/pro‑proliferative processes in human acute myeloid leukemia cells.

Sphingosine-1-phosphate receptor 1 (S1PR1), the Gprotein-coupled receptor1 of sphingosine 1-phosphate (S1P), is crucial in the progression of various types of solid tumor through the positive regulation of anti‑apoptotic/pro‑survival responses by S1P‑S1PR1 signaling. However, the role of S1PR1 in the progression of human acute myeloid leukemia (AML) remains to be fully elucidated. In the present study, the correlation between the expression level of S1PR1 and the growth and apoptosis of human AML cells was investigated. Using overexpression and RNA interference knockdown assays, it was shown that the expression of S1PR1 in several human myeloid leukemia cell lines contributed to the suppression of cell apoptosis and promotion of cell proliferation. S1PR1 inhibited the expression level of Bcell lymphoma‑2‑associatedXprotein, a mitochondrion‑associated apoptosis promoter, and prevented caspase‑3 cleavage, indicating that S1PR1 interfered with the mitochondrion‑associated apoptotic processes in AML cells. In addition, the present study demonstrated that S1PR1 suppressed the generation of reactive oxygen species (ROS) in AML cells, and that the apoptosis mediated by the downregulation of S1PR1 was partially reversed by treatment with N‑acetyl‑L‑cysteine, a ROS scavenging agent, suggesting that the S1PR1‑induced resistance of cell apoptosis resulted, at least partially, from the suppression of ROS generation. Additionally, S1PR1 was able to interfere with the signaling of c‑Jun N‑terminal kinase, which may have also contribute dto the suppression of cell apoptosis. By contrast, extracellular signal‑regulated kinase (ERK) signaling appeared to be positively modulated by the S1PR1‑induced enhancement of mitogen‑activated protein kinase (MAPK) kinase1 (MKK1) activation, suggesting that S1PR1 may promote cell survival and proliferation through enhancing the activation of MKK1‑ERK signaling. In conclusion, S1PR1 likely serves as an anti‑apoptotic/pro‑proliferative protein in AML cells through the inhibition of mitochondrion‑associated apoptosis and ROS generation, and via the regulation of multiple MAPK signaling cascades.

MASM, a Matrine Derivative, Offers Radioprotection by Modulating Lethal Total-Body Irradiation-Induced Multiple Signaling Pathways in Wistar Rats.

Matrine is an alkaloid extracted from Sophora flavescens Ait and has many biological activities, such as anti-inflammatory, antitumor, anti-fibrosis, and immunosuppressive properties. In our previous studies, the matrine derivative MASM was synthesized and exhibited potent inhibitory activity against liver fibrosis. In this study, we mainly investigated its protection against lethal total-body irradiation (TBI) in rats. Administration of MASM reduced the radiation sickness characteristics and increased the 30-day survival of rats before or after lethal TBI. Ultrastructural observation illustrated that pretreatment of rats with MASM significantly attenuated the TBI-induced morphological changes in the different organs of irradiated rats. Gene expression profiles revealed that pretreatment with MASM had a dramatic effect on gene expression changes caused by TBI. Pretreatment with MASM prevented differential expression of 53% (765 genes) of 1445 differentially expressed genes induced by TBI. Pathway enrichment analysis indicated that these genes were mainly involved in a total of 21 pathways, such as metabolic pathways, pathways in cancer, and mitogen-activated protein kinase (MAPK) pathways. Our data indicated that pretreatment of rats with MASM modulated these pathways induced by TBI, suggesting that the pretreatment with MASM might provide the protective effects on lethal TBI mainly or partially through the modulation of these pathways, such as multiple MAPK pathways. Therefore, MASM has the potential to be used as an effective therapeutic or radioprotective agent to minimize irradiation damages and in combination with radiotherapy to improve the efficacy of cancer therapy.

Overcoming MITF-conferred drug resistance through dual AURKA/MAPK targeting in human melanoma cells.

MITF (microphthalmia-associated transcription factor) is a frequently amplified lineage-specific oncogene in human melanoma, whose role in intrinsic drug resistance has not been systematically investigated. Utilizing chemical inhibitors for major signaling pathways/cellular processes, we witness MITF as an elicitor of intrinsic drug resistance. To search kinase(s) targets able to bypass MITF-conferred drug resistance, we employed a multi-kinase inhibitor-directed chemical proteomics-based differential affinity screen in human melanocytes carrying ectopic MITF overexpression. A subsequent methodical interrogation informed mitotic Ser/Thr kinase Aurora Kinase A (AURKA) as a crucial regulator of melanoma cell proliferation and migration, independent of the underlying molecular alterations, including TP53 functional status and MITF levels. Crucially, assessing the efficacy of investigational AURKA inhibitor MLN8237, we pre-emptively witness the procurement of a molecular program consistent with acquired drug resistance. This involved induction of multiple MAPK (mitogen-activated protein kinase) signaling pathway components and their downstream proliferation effectors (Cyclin D1 and c-JUN) and apoptotic regulators (MITF and Bcl-2). A concomitant AURKA/BRAF and AURKA/MEK targeting overcame MAPK signaling activation-associated resistance signature in BRAF- and NRAS-mutated melanomas, respectively, and elicited heightened anti-proliferative activity and apoptotic cell death. These findings reveal a previously unreported MAPK signaling-mediated mechanism of immediate resistance to AURKA inhibitors. These findings could bear significant implications for the application and the success of anti-AURKA approaches that have already entered phase-II clinical trials for human melanoma.

Genome-wide identification and transcriptional expression analysis of mitogen-activated protein kinase and mitogen-activated protein kinase kinase genes in Capsicum annuum.

The tripartite mitogen-activated protein kinase (MAPK) signaling cascades have been implicated in plant growth, development, and environment adaptation, but a comprehensive understanding of MAPK signaling at genome-wide level is limited in Capsicum annuum. Herein, genome-wide identification and transcriptional expression analysis of MAPK and MAPK kinase (MAPKK) were performed in pepper. A total of 19 pepper MAPK (CaMAPKs) genes and five MAPKK (CaMAPKKs) genes were identified. Phylogenetic analysis indicated that CaMAPKs and CaMAPKKs could be classified into four groups and each group contains similar exon-intron structures. However, significant divergences were also found. Notably, five members of the pepper MAPKK family were much less conserved than those found in Arabidopsis, and 9 Arabidopsis MAPKs did not have orthologs in pepper. Additionally, 7 MAPKs in Arabidopsis had either two or three orthologs in the pepper genome, and six pepper MAPKs and one MAPKK differing in sequence were found in three pepper varieties. Quantitative real-time RT-PCR analysis showed that the majority of MAPK and MAPKK genes were ubiquitously expressed and transcriptionally modified in pepper leaves after treatments with heat, salt, and Ralstonia solanacearum inoculation as well as exogenously applied salicylic acid, methyl jasmonate, ethephon, and abscisic acid. The MAPKK-MAPK interactome was tested by yeast two-hybrid assay, the results showed that one MAPKK might interact with multiple MAPKs, one MAPK might also interact with more than one MAPKKs, constituting MAPK signaling networks which may collaborate in transmitting upstream signals into appropriate downstream cellular responses and processes. These results will facilitate future functional characterization of MAPK cascades in pepper.

Protective Effects of Hong Shan Capsule against Lethal Total-Body Irradiation-Induced Damage in Wistar Rats.

Hong Shan Capsule (HSC), a crude drug of 11 medicinal herbs, was used in clinical practice for the treatment of radiation injuries in China. In this study, we investigated its protection in rats against acute lethal total-body irradiation (TBI). Pre-administration of HSC reduced the radiation sickness characteristics, while increasing the 30-day survival of the irradiated rats. Administration of HSC also reduced the radiation sickness characteristics and increased the 30-day survival of mice after exposure to lethal TBI. Ultrastructural observation illustrated that the pretreatment of rats with HSC significantly attenuated the TBI-induced morphological changes in the different organs of irradiated rats. Gene expression profiles revealed the dramatic effect of HSC on alterations of gene expression caused by lethal TBI. Pretreatment with HSC prevented differential expression of 66% (1398 genes) of 2126 genes differentially expressed in response to TBI. Pathway enrichment analysis indicated that these genes were mainly involved in a total of 32 pathways, such as pathways in cancer and the mitogen-activated protein kinase (MAPK) signaling pathway. Our analysis indicated that the pretreatment of rats with HSC modulated these pathways induced by lethal TBI, such as multiple MAPK pathways, suggesting that pretreatment with HSC might provide protective effects on lethal TBI mainly or partially through the modulation of these pathways. Our data suggest that HSC has the potential to be used as an effective therapeutic or radio-protective agent to minimize irradiation damage.

ERK 1/2 Mediates H2O2‐Induced Increase in Renal Epithelial Paracellular Permeability

The paracellular permeability of renal epithelia is determined primarily by the integrity of the tight junction (TJ) structure located at the apicolateral borders of adjacent epithelial cells. Treatment of confluent populations of MDCK cells (distal tubule‐like) and LLC‐PK1 cells (proximal tubule‐like) with hydrogen peroxide (H2O2) increases the paracellular permeability to large solutes in a concentration‐dependent manner. H2O2 was cytotoxic at higher concentrations. We have examined the involvement of Mitogen‐Activated Protein Kinases (MAPKs) in mediating these H2O2 actions. Treatment with H2O2 increased the phosphorylation (activation) of multiple MAPKs, ERK ½, JNK ½, and p38MAPK, in a time‐dependent manner. Phosphorylation of each MAPK peaked at 10‐30 minutes and declined by 120 minutes. Pretreatment of cells with U0126 or PD98059, inhibitors of ERK ½ activation, blocked the ability of H­2O2 to increase paracellular permeability. SB202190, a p38MAPK inhibitor, diminished but did not entirely block the H2O2 effect. Treatment of cells with SP600125, an inhibitor of JNK ½ activation, did not affect the ability of H2O2 to increase paracellular permeability. Pretreatment with U0126 and SB202190, but not SP600125, also diminished the cytotoxicity observed at higher H2O2 concentrations. These results indicate that MAPKs, in particular ERK ½, have a central role in mediating the effects of H2O2 on both modulation of paracellular permeability and induction of cytotoxicity in renal epithelial cells. (supported by NIH 1R15DK091749)

A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib

Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors.

Manipulation of mitogen-activated protein kinase kinase signaling in the Arabidopsis stomatal lineage reveals motifs that contribute to protein localization and signaling specificity.

When multiple mitogen-activated protein kinase (MAPK) components are recruited recurrently to transduce signals of different origins, and often opposing outcomes, mechanisms to enforce signaling specificity are of utmost importance. These mechanisms are largely uncharacterized in plant MAPK signaling networks. The Arabidopsis thaliana stomatal lineage was previously used to show that when rendered constitutively active, four MAPK kinases (MKKs), MKK4/5/7/9, are capable of perturbing stomatal development and that these kinases comprise two pairs, MKK4/5 and MKK7/9, with both overlapping and divergent functions. We characterized the contributions of specific structural domains of these four "stomatal" MKKs to MAPK signaling output and specificity both in vitro and in vivo within the three discrete cell types of the stomatal lineage. These results verify the influence of functional docking (D) domains of MKKs on MAPK signal output and identify novel regulatory functions for previously uncharacterized structures within the N termini of MKK4/5. Beyond this, we present a novel function of the D-domains of MKK7/9 in regulating the subcellular localization of these kinases. These results provide tools to broadly assess the extent to which these and additional motifs within MKKs function to regulate MAPK signal output throughout the plant.

Mixed-lineage kinase 3 deficiency promotes neointima formation through increased activation of the RhoA pathway in vascular smooth muscle cells.

Mitogen-activated protein kinase pathways play an important role in neointima formation secondary to vascular injury, in part by promoting proliferation of vascular smooth muscle cells (VSMC). Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase that activates multiple mitogen-activated protein kinase pathways and has been implicated in regulating proliferation in several cell types. However, the role of MLK3 in VSMC proliferation and neointima formation is unknown. The aim of this study was to determine the function of MLK3 in the development of neointimal hyperplasia and to elucidate the underlying mechanisms. Neointima formation was analyzed after endothelial denudation of carotid arteries from wild-type and MLK3-deficient mice. MLK3 deficiency promoted injury-induced neointima formation and increased proliferation of primary VSMC derived from aortas isolated from MLK3-deficient mice compared with wild-type mice. Furthermore, MLK3 deficiency increased the activation of p63Rho guanine nucleotide exchange factor, RhoA, and Rho kinase in VSMC, a pathway known to promote neointimal hyperplasia, and reconstitution of MLK3 expression attenuated Rho kinase activation. Furthermore, cJun NH2-terminal kinase activation was decreased in MLK3-deficient VSMC, and proliferation of wild-type but not MLK3 knockout cells treated with a cJun NH2-terminal kinase inhibitor was attenuated. We demonstrate that MLK3 limits RhoA activation and injury-induced neointima formation by binding to and inhibiting the activation of p63Rho guanine nucleotide exchange factor, a RhoA activator. In MLK3-deficient cells, activation of p63Rho guanine nucleotide exchange factor proceeds in an unchecked manner, leading to a net increase in RhoA pathway activation. Reconstitution of MLK3 expression restores MLK3/p63Rho guanine nucleotide exchange factor interaction, which is attenuated by feedback from activated cJun NH2-terminal kinase.

Combining docking site and phosphosite predictions to find new substrates: Identification of smoothelin-like-2 (SMTNL2) as a c-Jun N-terminal kinase (JNK) substrate

Specific docking interactions between mitogen-activated protein kinases (MAPKs), their regulators, and their downstream substrates, are crucial for efficient and accurate signal transmission. To identify novel substrates of the c-Jun N-terminal kinase (JNK) family of MAPKs, we searched the human genome for proteins that contained (1), a predicted JNK-docking site (D-site); and (2), a cluster of putative JNK target phosphosites located close to the D-site. Here we describe a novel JNK substrate that emerged from this analysis, the functionally uncharacterized protein smoothelin-like 2 (SMTNL2). SMTNL2 protein bound with high-affinity to multiple MAPKs including JNK1-3 and ERK2; furthermore, the identity of conserved amino acids in the predicted docking site (residues 180–193) was necessary for this high-affinity binding. In addition, purified full-length SMTNL2 protein was phosphorylated by JNK1-3 in vitro, and this required the integrity of the D-site. Using mass spectrometry and mutagenesis, we identified four D-site-dependent phosphoacceptor sites in close proximity to the docking site, at S217, S241, T236 and T239. A short peptide comprised of the SMTNL2 D-site inhibited JNK-mediated phosphorylation of the ATF2 transcription factor, showing that SMTNL2 can compete with other substrates for JNK binding. Moreover, when transfected into HEK293 cells, SMTNL2 was phosphorylated by endogenous JNK in a D-site dependent manner, on the same residues identified in vitro. SMTNL2 protein was expressed in many mammalian tissues, with a notably high expression in skeletal muscle. Consistent with the hypothesis that SMTNL2 has a function in skeletal muscle, SMTNL2 protein expression was strongly induced during the transition from myoblasts to myotubes in differentiating C2C12 cells.