Canonical Mitogen-activated Protein Kinase Research Articles

Abstract B063: Differential pathway engagement and the biological consequences of KRAS variants in cancer

Abstract KRAS is a proto-oncogene found mutated in >90% of pancreatic ductal adenocarcinomas (PDAC) and encodes for a GTPase that mediates signaling transduction through the mitogen-activated protein kinase (MAPK) and PI3K/AKT pathways. Mutations in amino acids G12, G13, and Q61 prevent its inactivation by regulatory GTPase-activating proteins (GAPs), resulting in its constitutive activation. Previous work indicates that KRAS mutants possess unique biochemical differences that could result in divergent allele-specific signaling networks, underscoring unique vulnerabilities and response to treatment. Furthermore, preclinical and clinical data reveal allele-specific differences in tumor initiation capacity and patient prognosis in PDAC. Therefore, dissecting the biochemical and biological differences among KRAS variants would allow the identification of new allele-specific strategies for therapy. We hypothesize that the biochemical differences between KRAS variants result in differential signaling pathway engagement, affecting cellular behavior and tumorigenic properties. To study the biology of KRAS mutant variants in PDAC, we used CRISPR-Cas9 to generate KRAS-deficient PDAC cell lines which survived KRAS ablation through adaptive and reversible signaling rewiring. Our initial observations suggested that, at comparable expression levels, KRAS mutants diverge in their baseline signaling through the canonical MAPK and PI3K pathways. Therefore, we leveraged this unique isogenic system to uncover allele-specific differences between KRAS variants in global gene expression (by RNA-seq) and phosphoproteomic signaling networks (by data-independent acquisition mass spectroscopy; DIA-MS). We further employed a multiplexed competitive fitness approach using a sequence-based readout to assess changes in cellular fitness in response to drug perturbations (KRASi, PI3Ki, SHP2i, ERKi, RTKi and MEKi). Our findings suggest that KRAS mutants cluster into groups that diverge in downstream pathway engagement and gene expression, leading to cluster-specific differences in drug sensitivity. Our findings suggest that elucidating the signaling differences between KRAS alleles and their biological functions could translate to novel therapeutic strategies in PDAC and other KRAS mutant cancers. Citation Format: Yanixa Quiñones Avilés, Jaffarguriqbal Singh, Wenxue Li, Yong Kong, Yansheng Liu, Mandar Deepak Muzumdar. Differential pathway engagement and the biological consequences of KRAS variants in cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B063.

Impact of Redox Modification on MAPK Global Substrate Selection

Mitogen activated protein kinases (MAPKs) are activated by various stimuli, ranging from growth factors to environmental stress factors. However, given this diverse set of stimuli, it is not currently clear how specificity is achieved in MAPK signaling pathways. We are exploring the hypothesis that, by modulating the substrate selectivity of MAPK family members, cells are able to control which “arm” of a branched pathway is activated in response to a given signal. Specifically we are investigating the impact of redox modification on the global substrate selection of the canonical MAPKs, extracellular regulated kinase 2 (ERK2) and p38α, which play important roles in regulating a variety of cellular outcomes, including cellular migration, differentiation, apoptosis, proliferation and survival. MAPK family members recognize the majority of their substrates via one of two binding surfaces, termed the D‐recognition site (DRS) and the F‐recognition site (FRS). Interestingly, MAPKs contain cysteine residues that are predicted to be oxidized by H2O2 that lie within or in close proximity to the substrate recognition sites. Therefore, to explore the effects of oxidation on MAPK substrate selection, we first investigated the ability of ERK2 and p38α to phosphorylate model DRS and FRS peptide substrates following treatment with various concentrations of H2O2. While their activity toward FRS substrates was not changed by H2O2 pretreatment, both ERK2 and p38α exhibited an increase in the relative activity toward the DRS substrate, Sub‐D, at H2O2 concentrations that were stoichiometric with the kinase. Kinetic analysis demonstrated that treatment with H2O2 leads to substantial changes in Km and, to a lesser extent, kcatfor both kinases. Together, these data suggest that redox modification of p38α and ERK2 may alter their ability to phosphorylate substrates recognized by the DRS. This raised the intriguing possibility that oxidation could alter the activity of p38α and ERK2 toward some, but not all, of their downstream substrates. To explore this possibility further, we investigated the impact of redox modification on the global substrate selectivity of these MAPKs using functional protein microarrays. Consistent with our hypothesis, phosphorylation of several substrates was unaffected by H2O2 treatment while others exhibited H2O2‐dependent changes in their phosphorylation status (both increases and decreases depending on the substrate). Likewise, fluorescence polarization assays suggest that oxidation of ERK2 increases its affinity for some ligands while decreasing its affinity for others. Interestingly, the upstream MAPK kinase, MEK1, also recognizes ERK1/2 via interactions with the DRS, suggesting that ERK1/2 activation dynamics may be affected by signal‐generated H2O2 within the cellular environment. Indeed, cell‐based assays suggested that pre‐treatment of NIH‐3T3 cells with the cell permeable H2O2 scavenger, PEG‐catalase, alters ERK1/2 phosphorylation following platelet‐derived growth factor (PDGF) treatment. Together, these findings suggest that redox modification of the cysteines located within the MAPK DRS alters the activation dynamics and may help to modulate downstream substrate selection of MAPK family members under physiological and pathological states.

Hemin-Induced Death Models Hemorrhagic Stroke and Is a Variant of Classical Neuronal Ferroptosis.

Ferroptosis is a caspase-independent, iron-dependent form of regulated necrosis extant in traumatic brain injury, Huntington disease, and hemorrhagic stroke. It can be activated by cystine deprivation leading to glutathione depletion, the insufficiency of the antioxidant glutathione peroxidase-4, and the hemolysis products hemoglobin and hemin. A cardinal feature of ferroptosis is extracellular signal-regulated kinase (ERK)1/2 activation culminating in its translocation to the nucleus. We have previously confirmed that the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor U0126 inhibits persistent ERK1/2 phosphorylation and ferroptosis. Here, we show that hemin exposure, a model of secondary injury in brain hemorrhage and ferroptosis, activated ERK1/2 in mouse neurons. Accordingly, MEK inhibitor U0126 protected against hemin-induced ferroptosis. Unexpectedly, U0126 prevented hemin-induced ferroptosis independent of its ability to inhibit ERK1/2 signaling. In contrast to classical ferroptosis in neurons or cancer cells, chemically diverse inhibitors of MEK did not block hemin-induced ferroptosis, nor did the forced expression of the ERK-selective MAP kinase phosphatase (MKP)3. We conclude that hemin or hemoglobin-induced ferroptosis, unlike glutathione depletion, is ERK1/2-independent. Together with recent studies, our findings suggest the existence of a novel subtype of neuronal ferroptosis relevant to bleeding in the brain that is 5-lipoxygenase-dependent, ERK-independent, and transcription-independent. Remarkably, our unbiased phosphoproteome analysis revealed dramatic differences in phosphorylation induced by two ferroptosis subtypes. As U0126 also reduced cell death and improved functional recovery after hemorrhagic stroke in male mice, our analysis also provides a template on which to build a search for U0126's effects in a variant of neuronal ferroptosis.SIGNIFICANCE STATEMENT Ferroptosis is an iron-dependent mechanism of regulated necrosis that has been linked to hemorrhagic stroke. Common features of ferroptotic death induced by diverse stimuli are the depletion of the antioxidant glutathione, production of lipoxygenase-dependent reactive lipids, sensitivity to iron chelation, and persistent activation of extracellular signal-regulated kinase (ERK) signaling. Unlike classical ferroptosis induced in neurons or cancer cells, here we show that ferroptosis induced by hemin is ERK-independent. Paradoxically, the canonical MAP kinase kinase (MEK) inhibitor U0126 blocks brain hemorrhage-induced death. Altogether, these data suggest that a variant of ferroptosis is unleashed in hemorrhagic stroke. We present the first, unbiased phosphoproteomic analysis of ferroptosis as a template on which to understand distinct paths to cell death that meet the definition of ferroptosis.

The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAF-MEK-ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signalingviaa direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports thedevelopment of novel MEK1/2 inhibitors that target the Custructural interface or blunt dedicated Cu delivery mechanismsviaCCS.

Nanoscale Zeolitic Imidazolate Framework-8 Activator of Canonical MAPK Signaling for Bone Repair.

Zeolitic imidazolate framework-8 (ZIF-8) is an important type of metal organic framework and has found numerous applications in the biomedical field. Our previous studies have demonstrated that nano ZIF-8-based titanium implants could promote osseointegration; however, its osteogenic capacity and the related mechanisms in bone regeneration have not been fully clarified. Presented here is a nanoscale ZIF-8 that could drive rat bone mesenchymal stem cell (rBMSC) differentiation into osteoblasts both in vitro and in vivo, and interestingly, nano ZIF-8 exhibited a better osteogenic effect compared with ionic conditions of Zn at the same concentration of Zn2+. Moreover, the cellular uptake mechanisms of the nanoparticles were thoroughly clarified. Specifically, nano ZIF-8 could enter the rBMSC cytoplasm probably via caveolae-mediated endocytosis and macropinocytosis. The intracellular and extracellular Zn2+ released from nano ZIF-8 and the receptors involved in the endocytosis may play a role in inducing activation of key osteogenic pathways. Furthermore, through transcriptome sequencing, multiple osteogenic pathways were found to be upregulated, among which nano ZIF-8 primarily phosphorylated ERK, thus activating the canonical mitogen-activated protein kinase pathway and promoting the osteogenesis of rBMSCs. Taken together, this study helps to elucidate the mechanism by which nano ZIF-8 regulates osteogenesis and suggests it to be a potential biomaterial for constructing multifunctional composites in bone tissue engineering.

BRAF Mutation in Colorectal Cancers: From Prognostic Marker to Targetable Mutation.

Simple SummaryColorectal cancer with a mutation in an oncogene BRAF has paid much attention, as it comprises a population with dismal prognosis since two decades ago. A series of research since then has successfully changed this malignancy to be treatable with specific treatment. Here we thoroughly overviewed the basic, translational and clinical studies on colorectal cancer with BRAF mutation from a physician’s viewpoint. Accumulating lines of evidence suggest that intervention of the trunk cellular growth signal transduction pathway, namely EGFR-RAS-RAF-MEK-ERK pathway, is a clue to controlling this disease. However, it is not so straightforward. Recent studies unveil the diverse and plastic nature of this signal transduction pathway. We will introduce our endeavor to conquer this condition, based on newly arriving datasets, and discuss how we could open the door to future development of CRC treatment. The Raf murine sarcoma viral oncogene homolog B (BRAF) mutation is detected in 8–12% of metastatic colorectal cancers (mCRCs) and is strongly correlated with poor prognosis. The recent success of the BEACON CRC study and the development of targeted therapy have led to the determination of BRAF-mutated mCRCs as an independent category. For nearly two decades, a growing body of evidence has established the significance of the BRAF mutation in the development of CRC. Herein, we overview both basic and clinical data relevant to BRAF-mutated CRC, mainly focusing on the development of treatment strategies. This review is organized into eight sections, including clinicopathological features, molecular features, prognosis, the predictive value of anti-epidermal growth factor receptor (EGFR) therapy, resistant mechanisms for BRAF-targeting treatment, the heterogeneity of the BRAF mutation, future perspectives, and conclusions. A characterization of the canonical mitogen-activated protein kinase (MAPK) pathway is essential for controlling this malignancy, and the optimal combination of multiple interventions for treatments remains a point of debate.

Structural and Molecular Determinants of CCS-Mediated Copper Activation of MEK1/2

Normal physiology relies on the precise coordination of intracellular signal transduction pathways that respond to nutrient availability to balance cell growth and cell death. We recently established a critical mechanistic function for the redox-active micronutrient copper (Cu) in the canonical mitogen activated protein kinase (MAPK) pathway at the level of MEK1 and MEK2. Here we report the X-ray crystal structure of Cu-MEK1 and reveal active site chemical ligands and oxidation state specificity for MEK1 Cu coordination. Mechanistically, the Cu chaperone CCS selectively bound to and facilitated Cu transfer to MEK1. Mutations in MEK1 that disrupt Cu(I) affinity or a CCS small molecule inhibitor reduced Cu-stimulated MEK1 kinase activity. These atomic and molecular level data provide the first mechanistic insights of Cu kinase signaling and could be exploited for the development of novel MEK1/2 inhibitors that either target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS.

Interleukin-17 Reduces βENaC via MAPK Signaling in Vascular Smooth Muscle Cells.

Degenerin proteins, such as the beta epithelial Na+ channel (βENaC), are essential in the intracellular signaling of pressure-induced constriction, an important vascular smooth muscle cell (VSMC) function. While certain cytokines reduce ENaC protein in epithelial tissue, it is unknown if interleukin-17 (IL-17), a potent pro-inflammatory cytokine, directly mediates changes in membrane-associated βENaC in VSMCs. Therefore, we tested the hypothesis that exposure to IL-17 reduces βENaC in VSMCs through canonical mitogen-activated protein kinase (MAPK) signaling pathways. We treated cultured rat VSMCs (A10 cell line) with IL-17 (1–100 ng/mL) for 15 min to 16 h and measured expression of βENaC, p38MAPK, c-jun kinase (JNK), and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). IL-17 reduced βENaC protein expression in a concentration-dependent fashion and increased phosphorylation of p38MAPK by 15 min and JNK by 8 h. NFκB was unaffected by IL-17 in VSMCs. IL-17 treatment reduced VSMC viability but had no effect on cell death. To determine the underlying signaling pathway involved in this response, VSMCs were treated before and during IL-17 exposure with p38MAPK or JNK inhibitors. We found that JNK blockade prevented IL-17-mediated βENaC protein suppression. These data demonstrate that the pro-inflammatory cytokine IL-17 regulates VSMC βENaC via canonical MAPK signaling pathways, raising the possibility that βENaC-mediated loss of VSMC function may occur in inflammatory disorders.

The MAPK substrate MASS proteins regulate stomatal development in Arabidopsis.

Stomata are specialized pores in the epidermis of the aerial parts of a plant, where stomatal guard cells close and open to regulate gas exchange with the atmosphere and restrict excessive water vapor from the plant. The production and patterning of the stomatal lineage cells in higher plants are influenced by the activities of the widely-used mitogen-activated protein kinase (MAPK) signaling components. The phenotype caused by the loss-of-function mutations suggested pivotal roles of the canonical MAPK pathway in the suppression of stomatal formation and regulation of stomatal patterning in Arabidopsis, whilst the cell type-specific manipulation of individual MAPK components revealed the existence of a positive impact on stomatal production. Among a large number of putative MAPK substrates in plants, the nuclear transcription factors SPEECHLESS (SPCH) and SCREAM (SCRM) are targets of MAPK 3 and 6 (MPK3/6) in the inhibition of stomatal formation. The polarity protein BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) is phosphorylated by MPK3/6 for localization and function in driving divisional asymmetries. Here, by functionally characterizing three MAPK SUBSTRATES IN THE STOMATAL LINEAGE (MASS) proteins, we establish that they are plasma membrane-associated, positive regulators of stomatal production. MPK6 can phosphorylate the MASS proteins in vitro and mutating the putative substrate sites interferes the subcellular partition and function of MASS in planta. Our fine-scale domain analyses identify critical subdomains of MASS2 required for specific subcellular localization and biological function, respectively. Furthermore, our data indicate that the MASS proteins may directly interact with the MAPKK Kinase YODA (YDA) at the plasma membrane. Thus, the deeply conserved MASS proteins are tightly connected with MAPK signaling in Arabidopsis to fine-tune stomatal production and patterning, providing a functional divergence of the YDA-MPK3/6 cascade in the regulation of plant developmental processes.

Impact of Redox Modification on MAPK Global Substrate Selection

Mitogen activated protein kinases (MAPKs) are activated by various stimuli, ranging from growth factors to environmental stress factors. However, given this diverse set of stimuli, it is not currently clear how specificity is achieved in MAPK signaling pathways. We are exploring the hypothesis that, by modulating the substrate selectivity of MAPK family members, cells are able to control which “arm” of a branched pathway is activated in response to a given signal. Specifically, we are investigating the impact of redox modification on the global substrate selection of the canonical MAPKs, extracellular regulated kinase 2 (ERK2) and p38α, which play important roles in regulating a variety of cellular outcomes, including cellular migration, differentiation, apoptosis, proliferation and survival. MAPK family members recognize the majority of their substrates via one of two binding surfaces, termed the D‐recognition site (DRS) and the F‐recognition site (FRS). Interestingly, MAPKs contain redox‐sensitive cysteine residues within or in close proximity to their substrate recognition sites. Therefore, to explore the effects of oxidation on MAPK substrate selection, we first investigated the ability of ERK2 and p38α to phosphorylate model DRS and FRS peptide substrates following pre‐treatment of the kinases with various concentrations of H2O2. While their activity toward FRS substrates was not changed by H2O2 pre‐treatment, both ERK2 and p38α exhibited an increase in their relative activity toward the DRS substrate, Sub‐D, at H2O2 concentrations that were stoichiometric with the kinase. Kinetic analysis demonstrated that treatment with H2O2 leads to substantial changes in Km and, to a lesser extent, kcat for both kinases. Together, these data suggest that redox modification of p38α and ERK2 may alter their ability to phosphorylate substrates recognized by the DRS. This raised the intriguing possibility that oxidation could alter the activity of p38α and ERK2 toward some, but not all, of their downstream substrates. To explore this possibility further, we investigated the impact of redox modification on the global substrate selectivity of these MAPKs using functional protein microarrays. Consistent with our hypothesis, phosphorylation of several substrates was unaffected by H2O2 treatment while others exhibited H2O2‐dependent changes in their phosphorylation status (both increases and decreases depending on the substrate). These findings suggest that redox modification of the cysteines located within the MAPK DRS may help to modulate downstream substrate selection of MAPK family members under physiological and pathological states.Support or Funding InformationThis research was supported by the NIH National Institute of General Medical Sciences through grants SC2GM113784 and SC1GM130545 (to RHN) and R01GM119227 (to LBP). Likewise, LM is supported through a Title III HBGI grant from the U.S. Department of Education.

Connectivity mapping of a chronic kidney disease progression signature identified lysine deacetylases as novel therapeutic targets

Tubulointerstitial injury is an important determinant of chronic kidney disease progression, yet treatment is limited. Accordingly, we derived a chronic kidney disease progression signature based on aging and disease in Col4a3-/- mice, a model associated with proteinuria and progressive loss of kidney function. Computational drug repurposing with the Connectivity Map identified vorinostat, a lysine deacetylase inhibitor, as a candidate treatment to reverse progression signature gene expression. Vorinostat administration significantly increased the lifespan of Col4a3-/- mice and attenuated tubulointerstitial fibrosis and JNK phosphorylation in the kidneys of Col4a3-/- mice. Invitro, vorinostat reduced albumin- and angiotensin II-induced activation of canonical mitogen-activated protein kinases in kidney tubular epithelial cells. Finally, a subset of murine progression signature genes was differentially expressed across kidney transcriptomic data from patients with focal segmental glomerulosclerosis, IgA nephropathy, and diabetic nephropathy. Thus, our findings suggest that lysine deacetylase inhibition may be a novel treatment to chronic kidney disease associated with proteinuria and progressive tubulointerstitial injury.

Rhodanthpyrone A and B play an anti-inflammatory role by suppressing the nuclear factor-κB pathway in macrophages.

Macrophage-associated inflammation is crucial for the pathogenesis of diverse diseases including metabolic disorders. Rhodanthpyrone (Rho) is an active component of Gentiana rhodantha, which has been used in traditional Chinese medicine to treat inflammation. Although synthesis procedures of RhoA and RhoB were reported, the biological effects of the specific compounds have never been explored. In this study, the anti-inflammatory activity and mechanisms of action of RhoA and RhoB were studied in lipopolysaccharide (LPS)-stimulated macrophages. Pretreatment with RhoA and RhoB decreased inducible nitric oxide synthase and cyclooxygenase-2 expressions in RAW 264.7 cells and in thioglycollate-elicited mouse peritoneal macrophages. In addition, it downregulated transcript levels of several inflammatory genes in LPS-stimulated RAW 264.7 cells, including inflammatory cytokines/chemokines (Tnfa, Il6, and Ccl2) and inflammatory mediators (Nos2 and Ptgs2). Macrophage chemotaxis was also inhibited by treatment with the compounds. Mechanistic studies revealed that RhoA and RhoB suppressed the nuclear factor (NF)-κB pathway, but not the canonical mitogen activated protein kinase pathway, in LPS-stimulated condition. Moreover, the inhibitory effect of RhoA and RhoB on inflammatory gene expressions was attenuated by treatment with an NF-κB inhibitor. Our findings suggest that RhoA and RhoB play an anti-inflammatory role at least in part by suppressing the NF-κB pathway during macrophage-mediated inflammation.

The localization and function of p38α mitogen-activated protein kinase in rat oocytes.

P38α mitogen-activated protein kinase (MAPK), which is a member of the canonical MAPK family, is activated in response to various extracellular stresses and plays a role in multiple cellular processes. In this study, we investigated the expression, subcellular localization and functional roles of p38α MAPK during the meiotic maturation of rat oocytes. We found that p38α MAPK phosphorylation (p-p38α MAPK, indicative of p38α MAPK activation) was low at the germinal vesicle (GV) stage, increased 3hr after germinal vesicle breakdown (GVBD) and maintained its maximum at metaphase I (MI) or metaphase II (MII). The p-p38α MAPK mainly accumulated in the GV and had no obvious expression in the nucleus. From GVBD to MII, p-p38α MAPK was distributed in the cytoplasm around either the chromosomes or the spindle. We used SB203580, an inhibitor of p38α MAPK, to investigate the possible functional role of p38α MAPK during rat oocyte meiotic maturation. Treatment of GV stage oocytes with 20μM SB203580 blocked p-p38α MAPK activity, and the spindles appeared abnormal. Additionally, the rate of GVBD after 3hr of culture with 20μM SB203580 (58.8%) was significantly inhibited compared with the control (82.5%, p<.05), and the polar body extrusion rate after 12hr of culture with SB203580 was also significantly decreased compared with the control (40.1% vs 73.3%, p<.05). Taken together, these data indicate that p38α MAPK may play a vital role in rat oocyte meiotic maturation.

Piconewton-Scale Analysis of Ras-BRaf Signal Transduction with Single-Molecule Force Spectroscopy.

Intermolecular interactions dominate the behavior of signal transduction in various physiological and pathological cell processes, yet assessing these interactions remains a challenging task. Here, this study reports a single-molecule force spectroscopic method that enables functional delineation of two interaction sites (≈35 pN and ≈90 pN) between signaling effectors Ras and BRaf in the canonical mitogen-activated protein kinase (MAPK) pathway. This analysis reveals mutations on BRaf at Q257 and A246, two sites frequently linked to cardio-faciocutaneous syndrome, result in ≈10-30 pN alterations in RasBRaf intermolecular binding force. The magnitude of changes in RasBRaf binding force correlates with the size of alterations in protein affinity and in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-sensitive glutamate receptor (-R)-mediated synaptic transmission in neurons expressing replacement BRaf mutants, and predicts the extent of learning impairments in animals expressing replacement BRaf mutants. These results establish single-molecule force spectroscopy as an effective platform for evaluating the piconewton-level interaction of signaling molecules and predicting the behavior outcome of signal transduction.

P53 loss does not permit escape from BrafV600E-induced senescence in a mouse model of lung cancer.

Lung cancer arises through the acquisition of a number of genetic lesions, with a preponderance of activating mutations in the canonical mitogen-activated protein kinase (MAPK) cascade (RTK-RAS-RAF-MEK). BrafV600E expression induces benign lung adenomas that fail to progress to adenocarcinoma because of oncogene-induced senescence (OIS). BrafV600E expression, coupled with simultaneous p53 ablation, permits bypass of senescence and progression to lung adenocarcinoma. However, spontaneous human tumors sustain mutations in a temporally separated manner. Here, we use a mouse lung cancer model where oncogene activation (BrafV600E expression) and tumor suppressor loss (p53 ablation) are independently controlled through the actions of Flp and Cre recombinase, respectively. We show that p53 loss before OIS is permissive for the transition from lung adenoma to adenocarcinoma. In contrast, p53 loss after senescence is established fails to enable escape from senescence and disease progression. This study demonstrates that BrafV600E induced senescence is irreversible in vivo and suggests that therapy-induced senescence would halt further tumor progression.

Structural Basis for the Subversion of MAP Kinase Signaling by an Intrinsically Disordered Parasite Secreted Agonist

SummaryThe causative agent of toxoplasmosis, the intracellular parasite Toxoplasma gondii, delivers a protein, GRA24, into the cells it infects that interacts with the mitogen-activated protein (MAP) kinase p38α (MAPK14), leading to activation and nuclear translocation of the host kinase and a subsequent inflammatory response that controls the progress of the parasite. The purification of a recombinant complex of GRA24 and human p38α has allowed the molecular basis of this activation to be determined. GRA24 is shown to be intrinsically disordered, binding two kinases that act independently, and is the only factor required to bypass the canonical mitogen-activated protein kinase activation pathway. An adapted kinase interaction motif (KIM) forms a highly stable complex that competes with cytoplasmic regulatory partners. In addition, the recombinant complex forms a powerful in vitro tool to evaluate the specificity and effectiveness of p38α inhibitors that have advanced to clinical trials, as it provides a hitherto unavailable stable and highly active form of p38α.

Phosphorylation of the Polarity Protein BASL Differentiates Asymmetric Cell Fate through MAPKs and SPCH

Cell polarization is commonly used for the regulation of stem cell asymmetric division in both animals and plants. Stomatal development in Arabidopsis, a process that produces breathing pores in the epidermis, requires asymmetric cell division to differentiate highly specialized guard cells while maintaining a stem cell population [1, 2]. The BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) protein exhibits a polarized localization pattern in the cell and is required for differential cell fates resulting from asymmetric cell division [3]. The polarization of BASL is made possible by a positive feedback loop with a canonical mitogen-activated protein kinase (MAPK) pathway that recruits the MAPKK kinase YODA (YDA) and MAPK 6 (MPK6) to the cortical polarity site [4]. Here, we study BASL intracellular dynamics and show that the membrane-associated BASL is slowly replenished at the cortical polarity site and that the mobility is tightly linked to its phosphorylation status. Because BASL polarity is only exhibited by one daughter cell after an asymmetric cell division, we study how BASL differentiallyfunctions in the two daughter cells. The YDA MAPKcascade transduces upstream ligand-receptor signaling [5-13] to the transcription factor SPEECHLESS (SPCH), which controls stomatal initiation and is directly suppressed by MPK3/6-mediated phosphorylation [14, 15]. We show that BASL polarization leads to elevated nuclear MPK6 signaling and lowered SPCH abundance in one of the two daughter cells. Therefore, two daughter cells are differentiated by BASL polarity-mediated differential suppression of SPCH, which may provide developmental plasticity in plant stem cell asymmetric cell division (ACD).

Epidermal Growth Factor Receptor Signaling to the Mitogen Activated Protein Kinase Pathway Bypasses Ras in Pancreatic Cancer Cells.

Epidermal growth factor (EGF) receptor (EGFR/HER1) is overexpressed in human pancreatic cancers. However, anti-EGFR therapy does not exhibit significant therapeutic activity with oncogenic K-ras mutation. We sought to assess the signaling relationship between EGFR and mutant K-ras, which is commonly detected in pancreatic cancer. Pancreatic cancer cells harboring mutated K-ras were treated with EGF to assess signaling from EGFR to mitogen-activated protein kinase (MAPK) pathway. The role of Ras family of proteins in transducing EGFR signals was assessed using short interfering RNA. Other components of MAPK and PI3K (phosphoinositide 3-kinase) pathways were examined for their roles in EGFR signaling. First, EGF signaling in pancreatic cancer cells occurs selectively through HER1. Second, knockdown of all Ras isoforms failed to block EGF-mediated phosphorylation of extracellular signal-regulated kinase (ERK). Inhibition of Raf was observed to partially abrogate ERK phosphorylation, whereas MEK inhibition resulted in complete attenuation of EGF-mediated ERK phosphorylation. Finally, inhibition of phosphoinositide 3-kinase/AKT and CDC42/PAK pathways did not block EGFR signaling. Our study results demonstrate that EGFR-mediated signaling in mutant K-ras pancreatic cancer cells does not follow canonical MAPK signaling. Our novel findings suggest the existence of alternate signaling pathways to downstream MAPK in the presence of mutant K-ras.

Genome-wide analysis of the barley MAPK gene family and its expression patterns in relation to Puccinia hordei infection

Mitogen-activated protein kinases (MAPKs) have been shown to act as key regulators of stress responses in model plant and crop species. So far, however, the MAPK family has not been systematically studied in barley. Herein, we identified 16 HvMAPKs (Hv—Hordeum vulgare) based on computational analysis of barley transcriptomics and genomics databases. HvMAPKs contain all canonical MAPK domains, except for HvMPK2, which lacks a MAPK domain signature. In addition, five HvMAPKs harbor TEY and ten HvMAPKs harbor TDY dual phosphorylation motif in the activation loop. Interestingly, HvMPK2 contains a MEY instead of TEY phosphorylation motif. We classified HvMAPKs into four major plant MAPK clades based on phylogeny reconstruction and anchored all HvMAPK genes to five out of seven barley chromosomes. Furthermore, we inoculated seedlings of susceptible barley line L94 and its isolines L94-Rph3 and L94-Rph7 with rust fungus Pucciniahordei and analyzed the expression of 16 HvMAPK genes using qRT-PCR at 1–4.5 days post inoculation. In total, six HvMAPK genes exhibited significantly altered expression by P. hordei infection. The expression of HvMPK5, HvMPK6, HvMPK7 and HvMPK12 (set one genes) was strongly induced especially during effector-triggered immunity (ETI), whereas the expression of HvMPK2 and HvMPK17 (set two genes) was specifically downregulated during ETI. Yet the expression of HvMPK8 was also specifically but weakly downregulated during ETI. Overall, the expression patterns suggest that set one genes positively regulate ETI in barley–P. hordei pathosystem, whereas set two genes negatively regulate ETI and/or programmed cell death in this pathosystem.

Identification and validation of dysregulated MAPK7 (ERK5) as a novel oncogenic target in squamous cell lung and esophageal carcinoma.

BackgroundMAPK7/ERK5 (extracellular-signal-regulated kinase 5) functions within a canonical three-tiered MAPK (mitogen activated protein kinase) signaling cascade comprising MEK (MAPK/ERK kinase) 5, MEKK(MEK kinase) 2/3 and ERK5 itself. Despite being the least well studied of the MAPK-modules, evidence supports a role for MAPK7-signaling in the pathology of several cancer types.Methods and resultsFluorescence in situ hybridization (FISH) analysis identified MAPK7 gene amplification in 4 % (3/74) of non-small cell lung cancers (NSCLC) (enriched to 6 % (3/49) in squamous cell carcinoma) and 2 % (2/95) of squamous esophageal cancers (sqEC). Immunohistochemical (IHC) analysis revealed a good correlation between MAPK7 gene amplification and protein expression. MAPK7 was validated as a proliferative oncogenic driver by performing in vitro siRNA knockdown of MAPK7 in tumor cell lines. Finally, a novel MEK5/MAPK7 co-transfected HEK293 cell line was developed and used for routine cell-based pharmacodynamic screening. Phosphorylation antibody microarray analysis also identified novel downstream pharmacodynamic (PD) biomarkers of MAPK7 kinase inhibition in tumor cells (pMEF2A and pMEF2D).ConclusionsTogether, these data highlight a broader role for dysregulated MAPK7 in driving tumorigenesis within niche populations of highly prevalent tumor types, and describe current efforts in establishing a robust drug discovery screening cascade.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1455-y) contains supplementary material, which is available to authorized users.