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An abstract of the article "The MEK1-ERK2 signaling pathway protects cardiomyocytes from ischemic damage in vivo," by D. J. Lips et al., is presented.

Abstract The Ras-dependent Raf/MEK/ERK1/2 mitogen-activated protein (MAP) kinase signaling pathway is a major regulator of cell proliferation and survival. Not surprisingly, hyperactivation of this pathway is frequently observed in human malignancies as a result of aberrant activation of receptor tyrosine kinases or gain-of-function mutations in RAS or RAF genes. Components of the ERK1/2 pathway are therefore viewed as attractive candidates for the development of targeted therapies of cancer. In this article, we briefly review the basic research that has laid the groundwork for the clinical development of small molecules inhibitors of the ERK1/2 pathway. We then present the current state of clinical evaluation of MEK1/2 inhibitors in cancer and discuss challenges ahead.

Abstract Background Viral infection and neoplastic transformation trigger endoplasmic reticulum (ER) stress. Thus, a large proportion of the cells that must be recognized by the immune system are stressed cells. Cells respond to ER stress by launching the unfolded protein response (UPR). The UPR regulates the two key processes that control major histocompatibility complex class I (MHC I)-peptide presentation: protein synthesis and degradation. We therefore asked whether and how the UPR impinges on MHC I-peptide presentation. Results We evaluated the impact of the UPR on global MHC I expression and on presentation of the H2Kb-associated SIINFEKL peptide. EL4 cells stably transfected with vectors coding hen egg lysozyme (HEL)-SIINFEKL protein variants were stressed with palmitate or exposed to glucose deprivation. UPR decreased surface expression of MHC I but did not affect MHC I mRNA level nor the total amount of intracellular MHC I proteins. Impaired MHC I-peptide presentation was due mainly to reduced supply of peptides owing to an inhibition of overall protein synthesis. Consequently, generation of H2Kb-SIINFEKL complexes was curtailed during ER stress, illustrating how generation of MHC I peptide ligands is tightly coupled to ongoing protein synthesis. Notably, the UPR-induced decline of MHC I-peptide presentation was more severe when the protein source of peptides was localized in the cytosol than in the ER. This difference was not due to changes in the translation rates of the precursor proteins but to increased stability of the cytosolic protein during ER stress. Conclusion Our results demonstrate that ER stress impairs MHC I-peptide presentation, and that it differentially regulates expression of ER- vs. cytosol-derived peptides. Furthermore, this work illustrates how ER stress, a typical feature of infected and malignant cells, can impinge on cues for adaptive immune recognition.

Abstract Background The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established. Methods Wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We studied the impact of MEK1 and MEK2 activation on cellular morphology, cell proliferation, survival, migration, invasiveness, and tumorigenesis in mice. RNA interference was used to test the requirement for MEK1 and MEK2 function in maintaining the proliferation of human colorectal cancer cells. Results We found that expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Mechanistically, activation of MEK1 or MEK2 up-regulates the expression of matrix metalloproteinases, promotes invasiveness and protects cells from undergoing anoikis. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect. Conclusion MEK1 and MEK2 isoforms have similar transforming properties and are able to induce the formation of metastatic intestinal tumors in mice. Our results suggest that MEK2 plays a more important role than MEK1 in sustaining the proliferation of human colorectal cancer cells.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver malignancy and is a major cause of cancer-related mortality in the world. This study aimed to characterize glutamine amino acid transporter expression profiles in HCC compared to those of normal liver cells. In vitro and in vivo models of HCC were studied using qPCR, whereas the prognostic significance of glutamine transporter expression levels within patient tumors was analyzed through RNAseq. Solute carrier (SLC) 1A5 and SLC38A2 were targeted through siRNA or gamma-p-nitroanilide (GPNA). HCC cells depended on exogenous glutamine for optimal survival and growth. Murine HCC cells showed superior glutamine uptake rate than normal hepatocytes ( p < 0.0001). HCC manifested a global reprogramming of glutamine transporters compared to normal liver: SLC38A3 levels decreased, whereas SLC38A1, SLC7A6, and SLC1A5 levels increased. Also, decreased SLC6A14 and SLC38A3 levels or increased SLC38A1, SLC7A6, and SLC1A5 levels predicted worse survival outcomes (all p < 0.05). Knockdown of SLC1A5 and/or SLC38A2 expression in human Huh7 and Hep3B HCC cells, as well as GPNA-mediated inhibition, significantly decreased the uptake of glutamine; combined SLC1A5 and SLC38A2 targeting had the most considerable impact (all p < 0.05). This study revealed glutamine transporter reprogramming as a novel hallmark of HCC and that such expression profiles are clinically significant.

Hepatocellular carcinoma (HCC) is a disease of high unmet medical need that has become a global health problem. The development of targeted therapies for HCC has been hindered by the incomplete understanding of HCC pathogenesis and the limited number of relevant preclinical animal models. We recently unveiled a previously uncharacterized YES kinase (encoded by YES1)-dependent oncogenic signaling pathway in HCC. To model this subset of HCC, we established a series of syngeneic cell lines from liver tumors of transgenic mice expressing activated human YES. The resulting cell lines (referred to as HepYF) were enriched for expression of stem cell and progenitor markers, proliferated rapidly, and were characterized by high SRC family kinase (SFK) activity and activated mitogenic signaling pathways. Transcriptomic analysis indicated that HepYF cells are representative of the most aggressive proliferation class G3 subgroup of HCC. HepYF cells formed rapidly growing metastatic tumors upon orthotopic implantation into syngeneic hosts. Treatment with sorafenib or the SFK inhibitor dasatinib markedly inhibited the growth of HepYF tumors. The new HepYF HCC cell lines provide relevant preclinical models to study the pathogenesis of HCC and test novel small-molecule inhibitor and immunotherapy approaches., Competing Interests: Competing interests S.M. reports research funding from Bristol-Myers Squibb through the Fonds d'accélération des collaborations en santé (FACS) of the Government of Quebec. The remaining authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Ambrosia artemisiifolia and Ambrosia trifida (Asteraceae) are important pest species and the two greatest sources of aeroallergens globally. Here, we took advantage of a hybrid to simplify genome assembly and present chromosome-level assemblies for both species. These assemblies show high levels of completeness with Benchmarking Universal Single-Copy Ortholog (BUSCO) scores of 94.5% for A. artemisiifolia and 96.1% for A. trifida and long terminal repeat (LTR) Assembly Index values of 26.6 and 23.6, respectively. The genomes were annotated using RNA data identifying 41,642 genes in A. artemisiifolia and 50,203 in A. trifida. More than half of the genome is composed of repetitive elements, with 62% in A. artemisiifolia and 69% in A. trifida. Single copies of herbicide resistance-associated genes PPX2L, HPPD, and ALS were found, while two copies of the EPSPS gene were identified; this latter observation may reveal a possible mechanism of resistance to the herbicide glyphosate. Ten of the 12 main allergenicity genes were also localized, some forming clusters with several copies, especially in A. artemisiifolia. The evolution of genome structure has differed among these two species. The genome of A. trifida has undergone greater rearrangement, possibly the result of chromoplexy. In contrast, the genome of A. artemisiifolia retains a structure that makes the allotetraploidization of the most recent common ancestor of the Heliantheae Alliance the clearest feature of its genome. When compared to other Heliantheae Alliance species, this allowed us to reconstruct the common ancestor's karyotype-a key step for furthering of our understanding of the evolution and diversification of this economically and allergenically important group., (© 2024 His Majesty the King in Right of Canada. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America. Reproduced with the permission of the Minister of Agriculture and Agri‐Food Canada.)

ERK3/MAPK6 activates MAP kinase-activated protein kinase (MK)-5 in selected cell types. Male MK5 haplodeficient mice show reduced hypertrophy and attenuated increase in Col1a1 mRNA in response to increased cardiac afterload. In addition, MK5 deficiency impairs cardiac fibroblast function. This study determined the effect of reduced ERK3 on cardiac hypertrophy following transverse aortic constriction (TAC) and fibroblast biology in male mice. Three weeks post-surgery, ERK3, but not ERK4 or p38α, co-immunoprecipitated with MK5 from both sham and TAC heart lysates. The increase in left ventricular mass and myocyte diameter was lower in TAC-ERK3 +/- than TAC-ERK3 +/+ hearts, whereas ERK3 haploinsufficiency did not alter systolic or diastolic function. Furthermore, the TAC-induced increase in Col1a1 mRNA abundance was diminished in ERK3 +/- hearts. ERK3 immunoreactivity was detected in atrial and ventricular fibroblasts but not myocytes. In both quiescent fibroblasts and "activated" myofibroblasts isolated from adult mouse heart, siRNA-mediated knockdown of ERK3 reduced the TGF-β-induced increase in Col1a1 mRNA. In addition, intracellular type 1 collagen immunoreactivity was reduced following ERK3 depletion in quiescent fibroblasts but not myofibroblasts. Finally, knocking down ERK3 impaired motility in both atrial and ventricular myofibroblasts. These results suggest that ERK3 plays an important role in multiple aspects of cardiac fibroblast biology., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Cytokinesis is the last step of cell division and is regulated by the small GTPase RhoA. RhoA activity is required for all steps of cytokinesis, including prior to abscission when daughter cells are ultimately physically separated. Like germ cells in all animals, the Caenorhabditis elegans embryonic germline founder cell initiates cytokinesis but does not complete abscission, leaving a stable intercellular bridge between the two daughter cells. Here, we identify and characterize C. elegans OSGN-1 as a cytokinetic regulator that promotes RhoA activity during late cytokinesis. Sequence analyses and biochemical reconstitutions reveal that OSGN-1 is a flavin-containing monooxygenase (MO). Genetic analyses indicate that the MO activity of OSGN-1 is required to maintain active RhoA at the end of cytokinesis in the germline founder cell and to stabilize the intercellular bridge. Deletion of OSGIN1 in human cells results in an increase in binucleation as a result of cytokinetic furrow regression, and this phenotype can be rescued by expressing a catalytically active form of C. elegans OSGN-1, indicating that OSGN-1 and OSGIN1 are functional orthologs. We propose that OSGN-1 and OSGIN1 are conserved MO enzymes required to maintain RhoA activity at the intercellular bridge during late cytokinesis and thus favor its stability, enabling proper abscission in human cells and bridge stabilization in C. elegans germ cells., Competing Interests: Competing interests statement:The authors declare no competing interest.

Extracellular signal-regulated kinase 3 (ERK3) is a poorly characterized member of the mitogen-activated protein (MAP) kinase family. Functional analysis of the ERK3 signaling pathway has been hampered by a lack of knowledge about the substrates and downstream effectors of the kinase. Here, we used large-scale quantitative phosphoproteomics and targeted gene silencing to identify direct ERK3 substrates and gain insight into its cellular functions. Detailed validation of one candidate substrate identified the gelsolin/villin family member supervillin (SVIL) as a bona fide ERK3 substrate. We show that ERK3 phosphorylates SVIL on Ser245 to regulate myosin II activation and cytokinesis completion in dividing cells. Depletion of SVIL or ERK3 leads to increased cytokinesis failure and multinucleation, a phenotype rescued by wild type SVIL but not by the non-phosphorylatable S245A mutant. Our results unveil a new function of the atypical MAP kinase ERK3 in cell division and the regulation of cell ploidy., (© 2022 Wiley Periodicals LLC.)

Most of our understanding regarding the involvement of SRC-family tyrosine kinases in cancer has stemmed from studies focused on the prototypical SRC oncogene. However, emerging research has shed light on the important role of YES signaling in oncogenic transformation, tumor growth, metastatic progression, and resistance to various cancer therapies. Clinical evidence indicates that dysregulated expression or activity of YES is a frequent occurrence in human cancers and is associated with unfavorable outcomes. These findings provide a compelling rationale for specifically targeting YES in certain cancer subtypes. Here, we review the crucial role of YES in cancer and discuss the challenges associated with translating preclinical observations into effective YES-targeted therapies., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Cell motility is a critical feature of invasive tumour cells that is governed by complex signal transduction events. Particularly, the underlying mechanisms that bridge extracellular stimuli to the molecular machinery driving motility remain partially understood. Here, we show that the scaffold protein CNK2 promotes cancer cell migration by coupling the pro-metastatic receptor tyrosine kinase AXL to downstream activation of ARF6 GTPase. Mechanistically, AXL signalling induces PI3K-dependent recruitment of CNK2 to the plasma membrane. In turn, CNK2 stimulates ARF6 by associating with cytohesin ARF GEFs and with a novel adaptor protein called SAMD12. ARF6-GTP then controls motile forces by coordinating the respective activation and inhibition of RAC1 and RHOA GTPases. Significantly, genetic ablation of CNK2 or SAMD12 reduces metastasis in a mouse xenograft model. Together, this work identifies CNK2 and its partner SAMD12 as key components of a novel pro-motility pathway in cancer cells, which could be targeted in metastasis., (© 2023. The Author(s).)

ERK3 and ERK4 define a distinct and understudied subfamily of mitogen-activated protein kinases (MAPKs). Little is known about the physiological roles of these atypical MAPKs and their association with human diseases. Interestingly, accumulating evidence points towards a role for ERK3 and ERK4 signaling in the initiation and progression of various types of cancer. Notably, a recent study reported that ERK4 is expressed in a subset of triple-negative breast cancer (TNBC) cell lines and that this expression is critical for AKT activation and for sustaining TNBC cell proliferation in vitro and tumor growth in mice. The authors also showed that depletion of ERK4 sensitizes TNBC cells to phosphatidylinositol-3-kinase (PI3K) inhibitors. They concluded that ERK4 is a promising therapeutic target for TNBC and has potential for combination therapy with PI3K inhibitors. Here, we raise concerns about the cellular models and experimental approaches used in this study, which compromise the conclusions on the oncogenic role of ERK4 in TNBC.

Melanoma is the deadliest form of skin cancer and considered intrinsically resistant to chemotherapy. Nearly all melanomas harbor mutations that activate the RAS/mitogen-activated protein kinase (MAPK) pathway, which contributes to drug resistance via poorly described mechanisms. Herein we show that the RAS/MAPK pathway regulates the activity of cyclin-dependent kinase 12 (CDK12), which is a transcriptional CDK required for genomic stability. We find that melanoma cells harbor constitutively high CDK12 activity, and that its inhibition decreases the expression of long genes containing multiple exons, including many genes involved in DNA repair. Conversely, our results show that CDK12 inhibition promotes the expression of short genes with few exons, including many growth-promoting genes regulated by the AP-1 and NF-κB transcription factors. Inhibition of these pathways strongly synergize with CDK12 inhibitors to suppress melanoma growth, suggesting promising drug combinations for more effective melanoma treatment., (© 2022. The Author(s).)

UBCH10 is an ubiquitin-conjugating enzyme (E2) of the anaphase-promoting complex E3 ligase, a key regulator of the cell cycle. The UBCH10 gene and protein are frequently upregulated in multiple solid tumors, associated with an unfavorable outcome. Accumulating evidence from studies of human cancer cell lines, mouse transgenic models, and analyses of clinical samples suggest that UBCH10 is a potential cancer drug target. No small molecule inhibitor of UBCH10 has been reported in the literature. Here, we described the development and optimization of a novel time-resolved fluorescence resonance energy transfer (TR-FRET) UBCH10 assay based on the self-polyubiquitination of the enzyme in the absence of E3. The homogenous assay is robust, sensitive, and scalable to different multi-well formats for high-throughput screening (HTS). We demonstrate the suitability of the TR-FRET assay to identify chemical inhibitors of UBCH10 in a pilot HTS campaign., Competing Interests: Declaration of competing interests The authors declare no potential conflicts of interests with respect to the research, authorship, and/or publication of this article., (Copyright © 2022. Published by Elsevier Inc.)

Identification of dominant, actionable oncogenic signaling pathways is key to guide the development of new targeted treatments for advanced-stage hepatocellular carcinoma (HCC). We have recently unveiled a novel YES-YAP/TAZ signaling axis involved in liver cancer development. Our study identifies the tyrosine kinase YES as a potential therapeutic target in HCC., Competing Interests: No potential conflicts of interest were reported by the author(s)., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The physiological functions and downstream effectors of the atypical mitogen-activated protein kinase extracellular signal-regulated kinase 3 (ERK3) remain to be characterized. We recently reported that mice expressing catalytically-inactive ERK3 (Mapk6 KD/KD ) exhibit a reduced postnatal growth rate as compared to control mice. Here, we show that genetic inactivation of ERK3 impairs postnatal skeletal muscle growth and adult muscle regeneration after injury. Loss of MAPK-activated protein kinase 5 (MK5) phenocopies the muscle phenotypes of Mapk6 KD/KD mice. At the cellular level, genetic or pharmacological inactivation of ERK3 or MK5 induces precocious differentiation of C2C12 or primary myoblasts, concomitant with MyoD activation. Reciprocally, ectopic expression of activated MK5 inhibits myogenic differentiation. Mechanistically, we show that MK5 directly phosphorylates FoxO3, promoting its degradation and reducing its association with MyoD. Depletion of FoxO3 rescues in part the premature differentiation of C2C12 myoblasts observed upon inactivation of ERK3 or MK5. Our findings reveal that ERK3 and its substrate MK5 act in a linear signaling pathway to control postnatal myogenic differentiation., (© 2022 Wiley Periodicals LLC.)

Most patients with hepatocellular carcinoma (HCC) are diagnosed at a late stage and have few therapeutic options and a poor prognosis. This is due to the lack of clearly defined underlying mechanisms or a dominant oncogene that can be targeted pharmacologically, unlike in other cancer types. Here, we report the identification of a previously uncharacterized oncogenic signaling pathway in HCC that is mediated by the tyrosine kinase Yes. Using genetic and pharmacological interventions in cellular and mouse models of HCC, we showed that Yes activity was necessary for HCC cell proliferation. Transgenic expression of activated Yes in mouse hepatocytes was sufficient to induce liver tumorigenesis. Yes phosphorylated the transcriptional coactivators YAP and TAZ (YAP/TAZ), promoting their nuclear accumulation and transcriptional activity in HCC cells and liver tumors. We also showed that YAP/TAZ were effectors of the Yes-dependent oncogenic transformation of hepatocytes. Src family kinase activation correlated with the tyrosine phosphorylation and nuclear localization of YAP in human HCC and was associated with increased tumor burden in mice. Specifically, high Yes activity predicted shorter overall survival in patients with HCC. Thus, our findings identify Yes as a potential therapeutic target in HCC.

Mitogen-activated protein kinase (MAPK) cascades are evolutionarily conserved signaling pathways that play essential roles in transducing extracellular environmental signals into diverse cellular responses to maintain homeostasis. These pathways are classically organized into an architecture of three sequentially acting protein kinases: a MAPK kinase kinase that phosphorylates and activates a MAPK kinase, which in turn phosphorylates and activates the effector MAPK. The activity of MAPKs is tightly regulated by phosphorylation of their activation loop, which can be modulated by positive and negative feedback mechanisms to control the amplitude and duration of the signal. The signaling outcomes of MAPK pathways are further regulated by interactions of MAPKs with scaffolding and regulatory proteins. Accumulating evidence indicates that, in addition to these mechanisms, MAPK signaling is commonly regulated by ubiquitin-proteasome system (UPS)-mediated control of the stability and abundance of MAPK pathway components. Notably, the biologic activity of some MAPKs appears to be regulated mainly at the level of protein turnover. Recent studies have started to explore the potential of targeted protein degradation as a powerful strategy to investigate the biologic functions of individual MAPK pathway components and as a new therapeutic approach to overcome resistance to current small-molecule kinase inhibitors. Here, we comprehensively review the mechanisms, physiologic importance, and pharmacological potential of UPS-mediated protein degradation in the control of MAPK signaling. SIGNIFICANCE STATEMENT: Accumulating evidence highlights the importance of targeted protein degradation by the ubiquitin-proteasome system in regulating and fine-tuning the signaling output of mitogen-activated protein kinase (MAPK) pathways. Manipulating protein levels of MAPK cascade components may provide a novel approach for the development of selective pharmacological tools and therapeutics., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Interleukin-17 receptor D (IL-17RD) is an evolutionarily conserved member of the IL-17 receptor family. Originally identified as a negative regulator of fibroblast growth factor (FGF) signaling under the name of Sef (Similar expression to FGF genes), IL-17RD was subsequently reported to regulate other receptor tyrosine kinase signaling pathways. In addition, recent studies have shown that IL-17RD also modulates IL-17 and Toll-like receptor (TLR) signaling. Combined genetic and cell biology studies have implicated IL-17RD in the control of cell proliferation and differentiation, cell survival, lineage specification, and inflammation. Accumulating evidence also suggest a role for IL-17RD in tumorigenesis. Expression of IL-17RD is down-regulated in various human cancers and recent work has shown that loss of IL-17RD promotes tumor formation in mice. However, the exact mechanisms underlying the tumor suppressor function of IL-17RD remain unclear and some studies have proposed that IL-17RD may exert pro-tumorigenic effects in certain contexts. Here, we provide an overview of the signaling functions of IL-17RD and review the evidence for its involvement in cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Girondel and Meloche.)

Interleukin-17 receptor D (IL-17RD), also known as similar expression to Fgf genes (SEF), is proposed to act as a signaling hub that negatively regulates mitogenic signaling pathways, like the ERK1/2 MAP kinase pathway, and innate immune signaling. The expression of IL-17RD is downregulated in certain solid tumors, which has led to the hypothesis that it may exert tumor suppressor functions. However, the role of IL-17RD in tumor biology remains to be studied in vivo. Here, we show that genetic disruption of Il17rd leads to the increased formation of spontaneous tumors in multiple tissues of aging mice. Loss of IL-17RD also promotes tumor development in a model of colitis-associated colorectal cancer, associated with an exacerbated inflammatory response. Colon tumors from IL-17RD-deficient mice are characterized by a strong enrichment in inflammation-related gene signatures, elevated expression of pro-inflammatory tumorigenic cytokines, such as IL-17A and IL-6, and increased STAT3 tyrosine phosphorylation. We further show that RNAi depletion of IL-17RD enhances Toll-like receptor and IL-17A signaling in colon adenocarcinoma cells. No change in the proliferation of normal or tumor intestinal epithelial cells was observed upon genetic inactivation of IL-17RD. Our findings establish IL-17RD as a tumor suppressor in mice and suggest that the protein exerts its function mainly by limiting the extent and duration of inflammation.

Despite its importance in human cancers, including colorectal cancers (CRC), oncogenic KRAS has been extremely challenging to target therapeutically. To identify potential vulnerabilities in KRAS-mutated CRC, we characterize the impact of oncogenic KRAS on the cell surface of intestinal epithelial cells. Here we show that oncogenic KRAS alters the expression of a myriad of cell-surface proteins implicated in diverse biological functions, and identify many potential surface-accessible therapeutic targets. Cell surface-based loss-of-function screens reveal that ATP7A, a copper-exporter upregulated by mutant KRAS, is essential for neoplastic growth. ATP7A is upregulated at the surface of KRAS-mutated CRC, and protects cells from excess copper-ion toxicity. We find that KRAS-mutated cells acquire copper via a non-canonical mechanism involving macropinocytosis, which appears to be required to support their growth. Together, these results indicate that copper bioavailability is a KRAS-selective vulnerability that could be exploited for the treatment of KRAS-mutated neoplasms.

Background: Due to the accessibility of underlying technologies the 'Omics', in particular genomics, are becoming commonplace in several fields of research, including the study of agricultural pests. The weed community is starting to embrace these approaches; genome sequences have been made available in the past years, with several other sequencing projects underway, as promoted by the International Weed Genome Consortium. Chromosome-scale sequences are essential to fully exploit the power of genetics and genomics., Results: We report such an assembly for Conyza canadensis, an important agricultural weed. Third-generation sequencing technology was used to create a genome assembly of 426 megabases, of which nine chromosome-scale scaffolds cover more than 98% of the entire assembled sequence. As this weed was the first to be identified with glyphosate resistance, and since we do not have a firm handle on the genetic mechanisms responsible for several herbicide resistances in the species, the genome sequence was annotated with genes known to be associated with herbicide resistance. A high number of ABC-type transporters, cytochrome P450 and glycosyltransferases (159, 352 and 181, respectively) were identified among the list of ab initio predicted genes., Conclusion: As C. canadensis has a small genome that is syntenic with other Asteraceaes, has a short life cycle and is relatively easy to cross, it has the potential to become a model weed species and, with the chromosome-scale genome sequence, contribute to a paradigm shift in the way non-target site resistance is studied. © 2020 Her Majesty the Queen in Right of CanadaPest Management Science © 2020 Society of Chemical Industry., (© 2020 Her Majesty the Queen in Right of CanadaPest Management Science © 2020 Society of Chemical Industry.)

The physiological functions of the atypical mitogen-activated protein kinase extracellular signal-regulated kinase 3 (ERK3) remain poorly characterized. Previous analysis of mice with a targeted insertion of the lacZ reporter in the Mapk6 locus ( Mapk6 lacZ ) showed that inactivation of ERK3 in Mapk6 lacZ mice leads to perinatal lethality associated with intrauterine growth restriction, defective lung maturation, and neuromuscular anomalies. To further explore the role of ERK3 in physiology and disease, we generated novel mouse models expressing a catalytically inactive ( Mapk6 KD ) or conditional ( Mapk6 Δ ) allele of ERK3. Surprisingly, we found that mice devoid of ERK3 kinase activity or expression survive the perinatal period without any observable lung or neuromuscular phenotype. ERK3 mutant mice reached adulthood, were fertile, and showed no apparent health problem. However, analysis of growth curves revealed that ERK3 kinase activity is necessary for optimal postnatal growth. To gain insight into the genetic basis underlying the discrepancy in phenotypes of different Mapk6 mutant mouse models, we analyzed the regulation of genes flanking the Mapk6 locus by quantitative PCR. We found that the expression of several Mapk6 neighboring genes is deregulated in Mapk6 lacZ mice but not in Mapk6 KD or Mapk6 Δ mutant mice. Our genetic analysis suggests that off-target effects of the targeting construct on local gene expression are responsible for the perinatal lethality phenotype of Mapk6 lacZ mutant mice., (Copyright © 2019 American Society for Microbiology.)

shRNA expression is an established technique to transiently or permanently deplete cells of a particular mRNA/protein. In functional analyses of oncogenic pathways it can thus be used as an alternative to pharmacologic inhibitors, or as a means to address otherwise undruggable targets. Here we describe and functionally validate a simple reiterative cloning system to generate concatenated multi-shRNA expression plasmids. The multi-shRNA expression cassette can eventually be subcloned into any suitably designed vector for the stable transfection of cells, here tested with derivatives of the Sleeping Beauty transposon vector for stable transfection of multiple myeloma cell lines at the lowest biosafety level. We finally test inducible versions of such multi-cassette knockdown vectors and show their efficacy for the induced concerted knockdown of all four components of the MEK/MAPK-module in the Ras/MAPK pathway. The described vector system(s) should be useful for functional knockdown analyses in a wide array of cell line models., Competing Interests: The authors have declared that no competing interests exist.

Recent work suggested that the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) is increased in the retinal pigment epithelium (RPE) of age-related macular degeneration (ARMD) patients and therefore could be an attractive therapeutic target. Notably, ERK1/2 pathway inhibitors are used in cancer therapy, with severe and noncharacterized ocular side effects. To decipher the role of ERK1/2 in RPE cells, we conditionally disrupted the Erk1 and Erk2 genes in mouse RPE. The loss of ERK1/2 activity resulted in a significant decrease in the level of RPE65 expression, a decrease in ocular retinoid levels concomitant with low visual function, and a rapid disorganization of RPE cells, ultimately leading to retinal degeneration. Our results identify the ERK1/2 pathway as a direct regulator of the visual cycle and a critical component of the viability of RPE and photoreceptor cells. Moreover, our results caution about the need for a very fine adjustment of kinase inhibition in cancer or ARMD treatment in order to avoid ocular side effects., (Copyright © 2017 Pyakurel et al.)

The RAS-RAF-MEK-ERK pathway has been intensively studied in oncology, with RAS known to be mutated in ∼30% of all human cancers. The recent emergence of ERK1/2 inhibitors and their ongoing clinical investigation demands a better understanding of ERK1/2 behavior following small-molecule inhibition. Although fluorescent fusion proteins and fluorescent antibodies are well-established methods of visualizing proteins, we show that ERK1/2 can be visualized via a less-invasive approach based on a two-step process using inverse electron demand Diels-Alder cycloaddition. Our previously reported trans-cyclooctene-tagged covalent ERK1/2 inhibitor was used in a series of imaging experiments following a click reaction with a tetrazine-tagged fluorescent dye. Although limitations were encountered with this approach, endogenous ERK1/2 was successfully imaged in cells, and "on-target" staining was confirmed by over-expressing DUSP5, a nuclear ERK1/2 phosphatase that anchors ERK1/2 in the nucleus.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) whose regulatory mechanisms and biological functions remain superficially understood. Contrary to most protein kinases, ERK3 is a highly unstable protein that is subject to dynamic regulation by the ubiquitin-proteasome system. However, the effectors that control ERK3 ubiquitination and degradation are unknown. In this study, we carried out an unbiased functional loss-of-function screen of the human deubiquitinating enzyme (DUB) family and identified ubiquitin-specific protease 20 (USP20) as a novel ERK3 regulator. USP20 interacts with and deubiquitinates ERK3 both in vitro and in intact cells. The overexpression of USP20 results in the stabilization and accumulation of the ERK3 protein, whereas USP20 depletion reduces the levels of ERK3. We found that the expression levels of ERK3 correlate with those of USP20 in various cellular contexts. Importantly, we show that USP20 regulates actin cytoskeleton organization and cell migration in a manner dependent on ERK3 expression. Our results identify USP20 as a bona fide regulator of ERK3 stability and physiological functions., (Copyright © 2017 American Society for Microbiology.)

Mouse embryonic stem (ES) cells have proven to be invaluable research tools for dissecting the role of signaling pathways in embryonic development, adult physiology, and various diseases. ES cells are amenable to genetic manipulation by classical gene targeting via homologous recombination or by genome editing technologies. These cells can be used to generate genetically modified mouse models or to study the signaling circuitry regulating self-renewal and early lineage commitment. In this chapter, we describe methods used for the isolation and establishment of mouse ES cell lines from blastocyst embryos and for the measurement of ERK1/2 activity in ES cells.

Background: Neonatal respiratory distress syndrome in preterm infants is a leading cause of neonatal death. Pulmonary insufficiency-related infant mortality rates have improved with antenatal glucocorticoid treatment and neonatal surfactant replacement. However, the mechanism of glucocorticoid-promoted fetal lung maturation is not understood fully, despite decades of clinical use. We previously have shown that genetic deletion of Erk3 in mice results in growth restriction, cyanosis, and early neonatal lethality because of pulmonary immaturity and respiratory distress. Recently, we demonstrated that the addition of postnatal surfactant administration to antenatal dexamethasone treatment resulted in enhanced survival of neonatal Erk3-null mice., Objective: To better understand the molecular underpinnings of corticosteroid-mediated lung maturation, we used high-throughput transcriptomic and high-resolution morphologic analysis of the murine fetal lung. We sought to examine the alterations in fetal lung structure and function that are associated with neonatal respiratory distress and antenatal glucocorticoid treatment., Study Design: Dexamethasone (0.4 mg/kg) or saline solution was administered to pregnant dams on embryonic days 16.5 and 17.5. Fetal lungs were collected and analyzed by microCT and RNA-seq for differential gene expression and pathway interactions with genotype and treatment. Results from transcriptomic analysis guided further investigation of candidate genes with the use of immunostaining in murine and human fetal lung tissue., Results: Erk3(-/-) mice exhibited atelectasis with decreased overall porosity and saccular space relative to wild type, which was ameliorated by glucocorticoid treatment. Of 596 differentially expressed genes (q < 0.05) that were detected by RNA-seq, pathway analysis revealed 36 genes (q < 0.05) interacting with dexamethasone, several with roles in lung development, which included corticotropin-releasing hormone and surfactant protein B. Corticotropin-releasing hormone protein was detected in wild-type and Erk3(-/-) lungs at E14.5, with significantly temporally altered expression through embryonic day 18.5. Antenatal dexamethasone attenuated corticotropin-releasing hormone at embryonic day 18.5 in both wild-type and Erk3(-/-) lungs (0.56-fold and 0.67-fold; P < .001). Wild type mice responded to glucocorticoid administration with increased pulmonary surfactant protein B (P = .003). In contrast, dexamethasone treatment in Erk3(-/-) mice resulted in decreased surfactant protein B (P = .012). In human validation studies, we confirmed that corticotropin-releasing hormone protein is present in the fetal lung at 18 weeks of gestation and increases in expression with progression towards viability (22 weeks of gestation; P < .01)., Conclusion: Characterization of whole transcriptome gene expression revealed glucocorticoid-mediated regulation of corticotropin-releasing hormone and surfactant protein B via Erk3-independent and -dependent mechanisms, respectively. We demonstrated for the first time the expression and temporal regulation of corticotropin-releasing hormone protein in midtrimester human fetal lung. This unique model allows the effects of corticosteroids on fetal pulmonary morphologic condition to be distinguished from functional gene pathway regulation. These findings implicate Erk3 as a potentially important molecular mediator of antenatal glucocorticoid action in promoting surfactant protein production in the preterm neonatal lung and expanding our understanding of key mechanisms of clinical therapy to improve neonatal survival., Competing Interests: The authors report no conflict of interest., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The protein kinases ERK1 and ERK2 are the effector components of the prototypical ERK1/2 mitogen-activated protein (MAP) kinase pathway. This signaling pathway regulates cell proliferation, differentiation and survival, and is essential for embryonic development and cellular homeostasis. ERK1 and ERK2 homologs share similar biochemical properties but whether they exert specific physiological functions or act redundantly has been a matter of controversy. However, recent studies now provide compelling evidence in support of functionally redundant roles of ERK1 and ERK2 in embryonic development and physiology. In this review, we present a critical assessment of the evidence for the functional specificity or redundancy of MAP kinase isoforms. We focus on the ERK1/ERK2 pathway but also discuss the case of JNK and p38 isoforms.

Aneuploidy is a common feature of human solid tumors and is often associated with poor prognosis. There is growing evidence that oncogenic signaling pathways, which are universally dysregulated in cancer, contribute to the promotion of aneuploidy. However, the mechanisms connecting signaling pathways to the execution of mitosis and cytokinesis are not well understood. Here, we show that hyperactivation of the ERK1/2 MAP kinase pathway in epithelial cells impairs cytokinesis, leading to polyploidization and aneuploidy. Mechanistically, deregulated ERK1/2 signaling specifically downregulates expression of the F-box protein Fbxw7β, a substrate-binding subunit of the SCF(Fbxw7) ubiquitin ligase, resulting in the accumulation of the mitotic kinase Aurora A. Reduction of Aurora A levels by RNA interference or pharmacological inhibition of MEK1/2 reverts the defect in cytokinesis and decreases the frequency of abnormal cell divisions induced by oncogenic H-Ras(V12). Reciprocally, overexpression of Aurora A or silencing of Fbxw7β phenocopies the effect of H-Ras(V12) on cell division. In vivo, conditional activation of MEK2 in the mouse intestine lowers Fbxw7β expression, resulting in the accumulation of cells with enlarged nuclei. We propose that the ERK1/2/ Fbxw7β/Aurora A axis identified in this study contributes to genomic instability and tumor progression.

In this study, we analyzed RNA-sequencing data of 14 samples characterized by biallelic CEBPA (CEBPA(bi)) mutations included in the Leucegene collection of 415 primary acute myeloid leukemia (AML) specimens, and describe for the first time high frequency recurrent mutations in the granulocyte colony-stimulating factor receptor gene CSF3R, which signals through JAK-STAT proteins. Chemical interrogation of these primary human specimens revealed a uniform and specific sensitivity to all JAK inhibitors tested irrespective of their CSF3R mutation status, indicating a general sensitization of JAK-STAT signaling in this leukemia subset. Altogether, these results identified the co-occurrence of mutations in CSF3R and CEBPA in a well-defined AML subset, which uniformly responds to JAK inhibitors and paves the way to personalized clinical trials for this disease., (© 2016 by The American Society of Hematology.)

Using next-generation sequencing of primary acute myeloid leukemia (AML) specimens, we identified to our knowledge the first unifying genetic network common to the two subgroups of KMT2A (MLL)-rearranged leukemia, namely having MLL fusions or partial tandem duplications. Within this network, we experimentally confirmed upregulation of the gene with the most subtype-specific increase in expression, LOC100289656, and identified cryptic MLL fusions, including a new MLL-ENAH fusion. We also identified a subset of MLL fusion specimens carrying mutations in SPI1 accompanied by inactivation of its transcriptional network, as well as frequent RAS pathway mutations, which sensitized the leukemias to synthetic lethal interactions between MEK and receptor tyrosine kinase inhibitors. This transcriptomics-based characterization and chemical interrogation of human MLL-rearranged AML was a valuable approach for identifying complementary features that define this disease.

ERK1 and ERK2 are the effector kinases of the ERK1/2 MAP-kinase signaling pathway, which plays a central role in transducing signals controlling cell proliferation, differentiation, and survival. Deregulated activity of the ERK1/2 pathway is linked to a group of developmental syndromes and contributes to the pathogenesis of various human diseases. One fundamental question that remains unaddressed is whether ERK1 and ERK2 have evolved unique physiological functions or whether they are used redundantly to reach a threshold of global ERK activity. Here, we show that the extent of development of the mouse placenta and embryo bearing different combinations of Erk1 and Erk2 alleles is strictly correlated with total ERK1/2 activity. We further demonstrate that transgenic expression of ERK1 fully rescues the embryonic and placental developmental defects associated with the loss of ERK2. We conclude that ERK1 and ERK2 exert redundant functions in mouse development., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Extracellular signal-regulated kinase 3 (ERK3 )is an atypical member of the mitogen-activated protein kinase (MAPK) family. We have previously shown that ERK3 is expressed during thymocyte differentiation and that its expression is induced in mature peripheral T cells following activation of ERK1/2 by T-cell receptor (TCR) signalling. Herein, we have investigated whether ERK3 expression is required for proper T-cell selection. Using a knock-in mouse model in which the coding sequence of ERK3 is replaced by the gene encoding for the β-galactosidase reporter, we show that ERK3 is expressed by double-positive (DP) thymocytes undergoing positive selection. In ERK3-deficient mice with a polyclonal TCR repertoire, we observe a decrease in positive selection. This reduction in positive selection was also observed when ERK3-deficient mice were backcrossed to class I- and class II-restricted TCR transgenic mice. Furthermore, the response of DP thymocytes to in vitro TCR stimulation was strongly reduced in ERK3-deficient mice. Together, these results show that ERK3 expression following TCR signalling is critical for proper thymic positive selection., (© 2014 John Wiley & Sons Ltd.)

It has been previously shown that the polycomb protein BMI1 and E4F1 interact physically and genetically in the hematopoietic system. Here, we report that E4f1 is essential for hematopoietic cell function and survival. E4f1 deletion induces acute bone marrow failure characterized by apoptosis of progenitors while stem cells are preserved. E4f1-deficient cells accumulate DNA damage and show defects in progression through S phase and mitosis, revealing a role for E4F1 in cell-cycle progression and genome integrity. Importantly, we showed that E4F1 interacts with and protects the checkpoint kinase 1 (CHK1) protein from degradation. Finally, defects observed in E4f1-deficient cells were fully reversed by ectopic expression of Chek1. Altogether, our results classify E4F1 as a master regulator of CHK1 activity that ensures high fidelity of DNA replication, thus safeguarding genome stability., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Multipotent long-term repopulating hematopoietic stem cells (LT-HSCs) can self-renew or differentiate into the less primitive short-term repopulating stem cells (ST-HSCs), which themselves produce progenitors that ensure the daily supply of all essential blood components. The Polycomb group (PcG) protein BMI1 is essential for the activity of both HSCs and progenitor cells. Although BMI1 operates by suppressing the Ink4a/Arf locus in progenitors and ST-HSCs, the mechanisms through which this gene regulates the activity of LT-HSCs remain poorly understood. Toward this goal, we isolated BMI1-containing protein complexes and identified UBAP2L as a novel BMI1-interacting protein. We also showed that UBAP2L is preferentially expressed in mouse and human HSC-enriched populations when compared with more mature cell types, and that this gene is essential for the activity of LT-HSCs. In contrast to what is observed for Bmi1 knockdown, we found that UBAP2L depletion does not affect the Ink4a/Arf locus. Given that we demonstrated that BMI1 overexpression is able to rescue the deleterious effects of Ubap2l downregulation on LT-HSC activity and that UBAP2L is part of a PcG subcomplex comprising BMI1, we propose a model in which at least 2 different BMI1-containing PcG complexes regulate HSC activity, which are distinguishable by the presence of UBAP2L., (© 2014 by The American Society of Hematology.)

Inhibition or allosteric modulation of mitogen-activated protein kinase kinases MEK1 and MEK2 (MEK1/2) represent a promising strategy for the discovery of new specific anticancer agents. In this paper, structure-based design, beginning from the lead compound PD98059, was used to study potential structural modifications on the chromone structure in order to obtain highly potent derivatives that target the allosteric pocket in MEK1. Subsequently, a small series of PD98059 analogs were synthesized to provide a first generation of chromone-based derivatives that inhibit the activation of MEK1 with IC50 values as low as 30 nM in vitro. Complementary cellular studies also showed that two of the compounds in the series inhibit the activity of MEK1/2 with IC50 values in the nanomolar range (73-97 nM). In addition, compounds in this series were found to inhibit the proliferation of a small panel of human cancer cell lines., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family whose function is largely unknown. Given the central role of MAPKs in T cell development, we hypothesized that ERK3 may regulate thymocyte development. Here we have shown that ERK3 deficiency leads to a 50% reduction in CD4(+) CD8(+) (DP) thymocyte number. Analysis of hematopoietic chimeras revealed that the reduction in DP thymocytes is intrinsic to hematopoietic cells. We found that early thymic progenitors seed the Erk3(-/-) thymus and can properly differentiate and proliferate to generate DP thymocytes. However, ERK3 deficiency results in a decrease in the DP thymocyte half-life, associated with a higher level of apoptosis. As a consequence, ERK3-deficient DP thymocytes are impaired in their ability to make successful secondary T cell receptor alpha (TCRα) gene rearrangement. Introduction of an already rearranged TCR transgene restores thymic cell number. We further show that knock-in of a catalytically inactive allele of Erk3 fails to rescue the loss of DP thymocytes. Our results uncover a unique role for ERK3, dependent on its kinase activity, during T cell development and show that this atypical MAPK is essential to sustain DP survival during RAG-mediated rearrangements., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: Respiratory distress syndrome (RDS) persists as a prevalent cause of infant morbidity and mortality. We have previously demonstrated that deletion of Erk3 results in pulmonary immaturity and neonatal lethality. Using RNA sequencing, we identified corticotrophin releasing hormone (CRH) and surfactant protein B (SFTPB) as potential molecular mediators of Erk3-dependent lung maturation. In this study, we characterized the impact of antenatal glucocorticoids and postnatal surfactant on neonatal survival of Erk3 null mice., Methods: In a double crossover design, we administered dexamethasone (dex) or saline to pregnant dams during the saccular stage of lung development, followed by postnatal surfactant or saline via inhalation intubation. Survival was recorded, and detailed lung histological analysis and staining for CRH and SFTPB protein expression were performed., Results: Without treatment, Erk3 null pups die within 6 h of birth with reduced aerated space, impaired thinning of the alveolar septa, and abundant glycogen stores, as described in human RDS. The administration of dex and surfactant improved RDS-associated lethality of Erk3(-/-) pups and partially restored functional fetal lung maturation by accelerating the downregulation of pulmonary CRH and partially rescuing the production of SFTPB., Conclusion: These findings emphasize that Erk3 is integral to terminal differentiation of type II cells, SFTPB production, and fetal pulmonary maturity.

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.

The extracellular signal-regulated kinase ERK1 and ERK2 (ERK1/2) cascade regulates a variety of cellular processes by phosphorylating multiple target proteins. The outcome of its activation ranges from stimulation of cell survival and proliferation to triggering tumor suppressor responses such as cell differentiation, cell senescence, and apoptosis. This pathway is intimately linked to cancer as several of its upstream activators are frequently mutated in human disease and are shown to accelerate tumorigenesis when engineered in the mouse genome. However, measurement of activated ERKs in human cancers or mouse models does not always support a role in tumorigenesis, and data consistent with a role in tumor suppression have been reported as well. The intensity of ERK signaling, negative feedback loops that regulate the pathway, and cross-talks with other signaling pathways, seem to be of primary importance in determining the final cellular outcome. Cell senescence, a putative tumor-suppression mechanism, depends on high-intensity ERK signals that trigger phosphorylation-dependent protein degradation of multiple proteins required for cell-cycle progression. This response may be circumvented during carcinogenesis by a variety of mechanisms, some of them yet to be discovered, which in essence turn ERK functions from tumor suppression to tumor promotion. The use of pharmacologic inhibitors targeting this pathway must be carefully evaluated so they are applied to cases in which ERKs are mainly oncogenic.

Oncogenic NRAS mutations are frequently identified in human myeloid leukemias. In mice, expression of endogenous oncogenic Nras (Nras(G12D/+)) in hematopoietic cells leads to expansion of myeloid progenitors, increased long-term reconstitution of bone marrow cells, and a chronic myeloproliferative neoplasm (MPN). However, acute expression of Nras(G12D/+) in a pure C57BL/6 background does not induce hyperactivated granulocyte macrophage colony-stimulating factor signaling or increased proliferation in myeloid progenitors. It is thus unclear how Nras(G12D/+) signaling promotes leukemogenesis. Here, we show that hematopoietic stem cells (HSCs) expressing Nras(G12D/+) serve as MPN-initiating cells. They undergo moderate hyperproliferation with increased self-renewal. The aberrant Nras(G12D/+) HSC function is associated with hyperactivation of ERK1/2 in HSCs. Conversely, downregulation of MEK/ERK by pharmacologic and genetic approaches attenuates the cycling of Nras(G12D/+) HSCs and prevents the expansion of Nras(G12D/+) HSCs and myeloid progenitors. Our data delineate critical mechanisms of oncogenic Nras signaling in HSC function and leukemogenesis.

The ERK1/2 MAP kinase pathway is an evolutionarily conserved signaling module that controls many fundamental physiological processes. Deregulated activity of ERK1/2 MAP kinases is associated with developmental syndromes and several human diseases. Despite the importance of this pathway, a comprehensive picture of the natural substrate repertoire and biochemical mechanisms regulated by ERK1/2 is still lacking. In this study, we used large-scale quantitative phosphoproteomics and bioinformatics analyses to identify novel candidate ERK1/2 substrates based on their phosphorylation signature and kinetic profiles in epithelial cells. We identified a total of 7936 phosphorylation sites within 1861 proteins, of which 155 classify as candidate ERK1/2 substrates, including 128 new targets. Candidate ERK1/2 substrates are involved in diverse cellular processes including transcriptional regulation, chromatin remodeling, RNA splicing, cytoskeleton dynamics, cellular junctions and cell signaling. Detailed characterization of one newly identified substrate, the transcriptional regulator JunB, revealed that ERK1/2 phosphorylate JunB on a serine adjacent to the DNA-binding domain, resulting in increased DNA-binding affinity and transcriptional activity. Our study expands the spectrum of cellular functions controlled by ERK1/2 kinases.

Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi-mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence.

We recently generated 2 phenotypically similar Hoxa9+Meis1 overexpressing acute myeloid leukemias that differ by their in vivo biologic behavior. The first leukemia, named FLA2, shows a high frequency of leukemia stem cells (LSCs; 1 in 1.4 cells), whereas the second, FLB1, is more typical with a frequency of LSCs in the range of 1 per several hundred cells. To gain insights into possible mechanisms that determine LSC self-renewal, we profiled and compared the abundance of nuclear and cytoplasmic proteins and phosphoproteins from these leukemias using quantitative proteomics. These analyses revealed differences in proteins associated with stem cell fate, including a hyperactive p38 MAP kinase in FLB1 and a differentially localized Polycomb group protein Ezh2, which is mostly nuclear in FLA2 and predominantly cytoplasmic in FLB1. Together, these newly documented proteomes and phosphoproteomes represent a unique resource with more than 440 differentially expressed proteins and 11 543 unique phosphopeptides, of which 80% are novel and 7% preferentially phosphorylated in the stem cell-enriched leukemia., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

Mitogen-activated protein kinase-activated protein (MAPKAP) kinase 5 (MK5) deficiency is associated with reduced extracellular signal-regulated kinase 3 (ERK3) (mitogen-activated protein kinase 6) levels, hence we utilized the MK5 knockout mouse model to analyze the physiological functions of the ERK3/MK5 signaling module. MK5-deficient mice displayed impaired dendritic spine formation in mouse hippocampal neurons in vivo. We performed large-scale interaction screens to understand the neuronal functions of the ERK3/MK5 pathway and identified septin7 (Sept7) as a novel interacting partner of ERK3. ERK3/MK5/Sept7 form a ternary complex, which can phosphorylate the Sept7 regulators Binders of Rho GTPases (Borgs). In addition, the brain-specific nucleotide exchange factor kalirin-7 (Kal7) was identified as an MK5 interaction partner and substrate protein. In transfected primary neurons, Sept7-dependent dendrite development and spine formation are stimulated by the ERK3/MK5 module. Thus, the regulation of neuronal morphogenesis is proposed as the first physiological function of the ERK3/MK5 signaling module.

Subcellular trafficking of key oncogenic signal pathway components is likely to be crucial for neoplastic transformation, but little is known about how such trafficking processes are spatially controlled. In this study, we show how Ras activation causes aberrant nuclear localization of phosphorylated mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK; MEK) MEK1/2 to drive neoplastic transformation. Phosphorylated MEK1/2 was aberrantly located within the nucleus of primary colorectal tumors and human colon cancer cells, and oncogenic activation of Ras was sufficient to induce nuclear accumulation of phosphorylated MEK1/2 and ERK1/2 in intestinal epithelial cells. Enforced nuclear localization of MEK1 in epithelial cells or fibroblasts was sufficient for hyperactivation of ERK1/2, thereby driving cell proliferation, chromosomal polyploidy, and tumorigenesis. Notably, Ras-induced nuclear accumulation of activated MEK1/2 was reliant on downregulation of the spatial regulator Sef, the reexpression of which was sufficient to restore normal MEK1/2 localization and a reversal of Ras-induced proliferation and tumorigenesis. Taken together, our findings indicate that Ras-induced downregulation of Sef is an early oncogenic event that contributes to genetic instability and tumor progression by sustaining nuclear ERK1/2 signaling., (©2012 AACR.)