Atypical Mitogen-activated Protein Kinase Research Articles

Role of the Atypical MAPK ERK3 in Cancer Growth and Progression.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) whose structural and regulatory features are distinct from those of conventional MAPKs, such as ERK1/2. Since its identification in 1991, the regulation, substrates and functions of ERK3 have remained largely unknown. However, recent years have witnessed a wealth of new findings about ERK3 signaling. Several important biological functions for ERK3 have been revealed, including its role in neuronal morphogenesis, inflammation, metabolism, endothelial cell tube formation and epithelial architecture. In addition, ERK3 has been recently shown to play important roles in cancer cell proliferation, migration, invasion and chemoresistance in multiple types of cancers. Furthermore, accumulating studies have uncovered various molecular mechanisms by which the expression level, protein stability and activity of ERK3 are regulated. In particular, several post-translational modifications (PTMs), including ubiquitination, hydroxylation and phosphorylation, have been shown to regulate the stability and activity of ERK3 protein. In this review, we discuss recent findings regarding biochemical and cellular functions of ERK3, with a main focus on its roles in cancers, as well as the molecular mechanisms of regulating its expression and activity.

The significance of ERK5 catalytic-independent functions in disease pathways.

Extracellular signal-regulated kinase 5 (ERK5), also known as BMK1 or MAPK7, represents a recent addition to the classical mitogen-activated protein kinase (MAPK) family. This family includes well-known members such as ERK1/2, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38 MAPK), as well as atypical MAPKs such as ERK3, ERK4, ERK7 (ERK8), and Nemo-like kinase (NLK). Comprehensive reviews available elsewhere provide detailed insights into ERK5, which interested readers can refer to for in-depth knowledge (Nithianandarajah-Jones et al., 2012; Monti et al., Cancers (Basel), 2022, 14). The primary aim of this review is to emphasize the essential characteristics of ERK5 and shed light on the intricate nature of its activation, with particular attention to the catalytic-independent functions in disease pathways.

Phosphoproteomic analysis identifies supervillin as an ERK3 substrate regulating cytokinesis and cell ploidy.

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.

On the Therapeutic Potential of ERK4 in Triple-Negative Breast Cancer.

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.

138 (PB128) - ERK3/MAPK6 has a dual role as pro-and anti-tumorigenic in Triple-Negative Breast Cancer

Extracellular-regulated kinase 3 (ERK3) - an atypical mitogen-activated protein kinase (MAPK), coded by the MAPK6 gene - is overexpressed in breast cancer (BC), strongly correlating with poor patient survival. However, the mechanisms by which ERK3 contributes to BC remain unknown. Therefore, this study aims to elucidate the role of ERK3 in breast cancer progression, especially in aggressive triple-negative BC subtype (TNBC).

An atypical MAPK regulates translocation of a GATA transcription factor in response to chemoattractant stimulation.

The Dictyostelium atypical mitogen-activated protein kinase (MAPK) Erk2 is required for chemotactic responses to cAMP as amoeba undergo multicellular development. In this study, Erk2 was found to be essential for the cAMP-stimulated translocation of the GATA transcription factor GtaC as indicated by the distribution of a GFP-GtaC reporter. Erk2 was also found to be essential for the translocation of GtaC in response to external folate, a foraging signal that directs the chemotaxis of amoeba to bacteria. Erk1, the only other Dictyostelium MAPK, was not required for the GtaC translocation to either chemoattractant, indicating that GFP-GtaC is a kinase translocation reporter specific for atypical MAPKs. The translocation of GFP-GtaC in response to folate was absent in mutants lacking the folate receptor Far1 or the coupled G-protein subunit Gα4. Loss of GtaC function resulted in enhanced chemotactic movement to folate, suggesting that GtaC suppresses responses to folate. The alteration of four Erk2-preferred phosphorylation sites in GtaC impacted the translocation of GFP-GtaC in response to folate and the GFP-GtaC-mediated rescue of aggregation and development of gtaC- cells. The ability of different chemoattractants to stimulate Erk2-regulated GtaC translocation suggests that atypical MAPK-mediated regulation of transcription factors can contribute to different cell fates.

DGKζ interacts with ERK3 and counteracts the promoting role of ERK3 in lung cancer migration

Extracellular‐Regulated Kinase 3 (ERK3) promotes cell migration and invasion in many cancers, including lung cancer. However, the mechanisms by which this atypical mitogen‐activated protein kinase (MAPK) is regulated remain poorly understood. In a yeast two‐hybrid assay, ERK3 was shown to interact with diacylglycerol kinase ζ (DGKζ), an enzyme which converts diacylglycerol to phosphatidic acid. This interaction has been confirmed by co‐immunoprecipitation and occurs through the C34 domain of ERK3 and the N‐terminus and C1 domain of DGKζ. By immunofluorescence, these proteins were shown to mainly co‐localize at protruding regions of the plasma membrane in cells. Interestingly, DGKζ reduces cell migration in lung cancer cell lines in a kinase‐independent manner. While ERK3 promotes cell motility in lung cancer cell lines, DGKζ overexpression negates this effect entirely in the transwell assay. Further, DGKζ reduces ERK3 accumulation at protrusions of the cell membrane but cannot alter localization of a mutant ERK3 lacking the C34 domain. In conclusion, we have identified DGKζ as a new interaction partner and inhibitor of ERK3 in regulating lung cancer cell migration.

MAPK-Activated Protein Kinases: Servant or Partner?

Mitogen-activated protein kinase (MAPK)-activated protein kinases (MAPKAPKs) are defined by their exclusive activation by MAPKs. They can be activated by classical and atypical MAPKs that have been stimulated by mitogens and various stresses. Genetic deletions of MAPKAPKs and availability of highly specific small-molecule inhibitors have continuously increased our functional understanding of these kinases. MAPKAPKs cooperate in the regulation of gene expression at the level of transcription; RNA processing, export, and stability; and protein synthesis. The diversity of stimuli for MAPK activation, the crosstalk between the different MAPKs and MAPKAPKs, and the specific substrate pattern of MAPKAPKs orchestrate immediate-early and inflammatory responses in space and time and ensure proper control of cell growth, differentiation, and cell behavior. Hence, MAPKAPKs are promising targets for cancer therapy and treatments for conditions of acute and chronic inflammation, such as cytokine storms and rheumatoid arthritis.

ERK3-MK5 signaling regulates myogenic differentiation and muscle regeneration by promoting FoxO3 degradation.

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 (Mapk6KD/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 Mapk6KD/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.

ERK3 is transcriptionally upregulated by \u2206Np63\u03b1 and mediates the role of \u2206Np63\u03b1 in suppressing cell migration in non-melanoma skin cancers

Backgroundp63, a member of the p53 gene family, is an important regulator for epithelial tissue growth and development. ∆Np63α is the main isoform of p63 and highly expressed in Non-melanoma skin cancer (NMSC). Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) whose biochemical features and cellular regulation are distinct from those of conventional MAPKs such as ERK1/2. While ERK3 has been shown to be upregulated in lung cancers and head and neck cancers, in which it promotes cancer cell migration and invasion, little is known about the implication of ERK3 in NMSCs.MethodsFluorescent immunohistochemistry was performed to evaluate the expression levels of ΔNp63α and ERK3 in normal and NMSC specimens. Dunnett’s test was performed to compare mean fluorescence intensity (MFI, indicator of expression levels) of p63 or ERK3 between normal cutaneous samples and NMSC samples. A mixed effects (ANOVA) test was used to determine the correlation between ΔNp63α and ERK3 expression levels (MFI). The regulation of ERK3 by ΔNp63α was studied by qRT-PCR, Western blot and luciferase assay. The effect of ERK3 regulation by ΔNp63α on cell migration was measured by performing trans-well migration assay.ResultsThe expression level of ∆Np63α is upregulated in NMSCs compared to normal tissue. ERK3 level is significantly upregulated in AK and SCC in comparison to normal tissue and there is a strong positive correlation between ∆Np63α and ERK3 expression in normal skin and skin specimens of patients with AK, SCC or BCC. Further, we found that ∆Np63α positively regulates ERK3 transcript and protein levels in A431 and HaCaT skin cells, underlying the upregulation of ERK3 expression and its positive correlation with ∆Np63α in NMSCs. Moreover, similar to the effect of ∆Np63α depletion, silencing ERK3 greatly enhanced A431 cell migration. Restoration of ERK3 expression under the condition of silencing ∆Np63α counteracted the increase in cell migration induced by the depletion of ∆Np63α. Mechanistically, ERK3 inhibits the phosphorylation of Rac1 G-protein and the formation of filopodia of A431 skin SCC cells.ConclusionsERK3 is positively regulated by ∆Np63α and mediates the role of ∆Np63α in suppressing cell migration in NMSC.

Dual Specific Phosphatase 14 Deletion Rescues Retinal Ganglion Cells and Optic Nerve Axons after Experimental Anterior Ischemic Optic Neuropathy

ABSTRACT Purpose Understanding molecular changes is essential for designing effective treatments for nonarteritic anterior ischemic optic neuropathy (AION), the most common acute optic neuropathy in adults older than 50 years. We investigated changes in the mitogen-activated protein kinase (MAPK) pathway after experimental AION and focused on dual specificity phosphatase 14 (Dusp14), an atypical MAPK phosphatase that is downstream of Krüppel-like transcription factor (KLF) 9-mediated inhibition of retinal ganglion cell (RGC) survival and axonal regeneration. Materials and methods We induced severe AION in a photochemical thrombosis model in adult C57BL/6 wild-type and Dusp14 knockout mice. For comparison, some studies were performed using an optic nerve crush model. We assessed changes in MAPK pathway molecules using Western blot and immunohistochemistry, measured retinal thickness using optical coherence tomography (OCT), and quantified RGCs and axons using histologic methods. Results Three days after severe AION, there was no change in the retinal protein levels of MAPK ERK1/2, phosphorylated-ERK1/2 (pERK1/2), downstream effector Elk-1 and phosphatase Dusp14 on Western blot. Western blot analysis of purified RGCs after a more severe model using optic nerve crush also showed no change in Dusp14 protein expression. Because of the known importance of the Dusp14 and MAPK pathway in RGCs, we examined changes after AION in Dusp14 knockout mice. Three days after AION, Dusp14 knockout mice had significantly increased pERK1/2+, Brn3A+ RGCs on immunohistochemistry. Three weeks after AION, Dusp14 knockout mice had significantly greater preservation of retinal thickness, increased number of Brn3A+ RGCs on whole mount preparation, and increased number of optic nerve axons compared with wild-type mice. Conclusions Genetic deletion of Dusp14, a MAPK phosphatase important in KFL9-mediated inhibition of RGC survival, led to increased activation of MAPK ERK1/2 and greater RGC and axonal survival after experimental AION. Inhibiting Dusp14 or activating the MAPK pathway should be examined further as a potential therapeutic approach to treatment of AION. Abbreviations: AION: anterior ischemic optic neuropathy; Dusp14: dual specific phosphatase 14; ERK1/2: extracellular signal–regulated kinases 1/2; Elk-1: ETS Like-1 protein; GCC: ganglion cell complex; GCL: ganglion cell layer; inner nuclear layer; KO: knockout; MAPK: mitogen-activated phosphokinase; OCT: optical coherence tomography; RGC: retinal ganglion cell; RNFL: retinal nerve fiber layer

Identification of atypical mitogen-activated protein kinase MAPK4 as a novel regulator in acute lung injury

BackgroundAcute lung injury (ALI) is a serious disease with highly morbidity and mortality that causes serious health problems worldwide. Atypical mitogen activated protein kinases (MAPKs) play critical roles in the development of tissues and have been proposed as promising therapeutic targets for various diseases. However, the potential role of atypical MAPKs in ALI remains elusive. In this study, we investigated the role of atypical MAPKs family member MAPK4 in ALI using LPS-induced murine ALI model.ResultsWe found that MAPK4 deficiency mice exhibited prolonged survival time after LPS challenge, accompanied by alleviated pathology in lung tissues, decreased levels of pro-inflammatory cytokines and altered composition of immune cells in BALF. Furthermore, the transduction of related signaling pathways, including MK5, AKT, JNK, and p38 MAPK pathways, was reduced obviously in LPS-treated MAPK4−/− mice. Notably, the expression of MAPK4 was up-regulated in lung tissues of ALI model, which was not related with MAPK4 promoter methylation, but negatively orchestrated by transcriptional factors NFKB1 and NR3C1. Further studies have shown that the expression of MAPK4 was also increased in LPS-treated macrophages. Meanwhile, MAPK4 deficiency reduced the expression of related pro-inflammatory cytokines in macrophage in response to LPS treatment. Finally, MAPK4 knockdown using shRNA pre-treatment could ameliorate the pathology of lung tissues and prolong the survival time of mice after LPS challenge.ConclusionsCollectively, these findings reveal an important biological function of atypical MAPK in mediating the pathology of ALI, indicating that MAPK4 might be a novel potential therapeutic target for ALI treatment.

Impact of Conventional and Atypical MAPKs on the Development of Metabolic Diseases.

The family of mitogen-activated protein kinases (MAPKs) consists of fourteen members and has been implicated in regulation of virtually all cellular processes. MAPKs are divided into two groups, conventional and atypical MAPKs. Conventional MAPKs are further classified into four sub-families: extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK1, 2 and 3), p38 (α, β, γ, δ), and extracellular signal-regulated kinase 5 (ERK5). Four kinases, extracellular signal-regulated kinase 3, 4, and 7 (ERK3, 4 and 7) as well as Nemo-like kinase (NLK) build a group of atypical MAPKs, which are activated by different upstream mechanisms than conventional MAPKs. Early studies identified JNK1/2 and ERK1/2 as well as p38α as a central mediators of inflammation-evoked insulin resistance. These kinases have been also implicated in the development of obesity and diabetes. Recently, other members of conventional MAPKs emerged as important mediators of liver, skeletal muscle, adipose tissue, and pancreatic β-cell metabolism. Moreover, latest studies indicate that atypical members of MAPK family play a central role in the regulation of adipose tissue function. In this review, we summarize early studies on conventional MAPKs as well as recent findings implicating previously ignored members of the MAPK family. Finally, we discuss the therapeutic potential of drugs targeting specific members of the MAPK family.

Abstract 474: ERK4-deficiency Potentiates the TAC-induced Increase in Collagen1-α 1 Messenger RNA in Mice

Cardiac hypertrophy, a common consequence of cardiopathologies such as hypertension and myocardial infarcts, involves formation of excessive interstitial fibrosis, which may impair cardiac function. Fibroblasts are the primary source of extracellular matrix protein. Extracellular-regulated kinase 4 (ERK4) is an atypical mitogen-activated protein kinase (MAPK). The regulation and role of ERK4 in the heart are currently unidentified and its only known target is MAP kinase-activated protein kinase 5 (MK5), a kinase involved in regulating fibroblast function. Following constriction of the transverse aorta (TAC), MK5 haplodeficient mice showed an attenuation of the TAC-induced increase in collagen 1-α 1 mRNA at 2-wk post-TAC and reduced hypertrophy 8-wk post-TAC. Further studies revealed MK5 immunoreactivity in cardiac fibroblasts but not myocytes. MK5 immunoprecipitates from whole heart contain ERK3 immunoreactivity, but not that of ERK4 or p38 MAPK. This study was to examine the role of ERK4 in myocardial structure, function, and remodeling 3-wk post-TAC. At 12 wk of age, echocardiographic imaging revealed systolic and diastolic function in male ERK4 -/- mice were similar to wild-type littermates (ERK4 +/+ ). Three weeks post-TAC, hypertrophy was similar in ERK4 +/+ and ERK4 -/- mice. Transcripts for BNP and βMHC increased to similar extent in TAC- ERK4 +/+ and TAC- ERK4 -/- mice. Two-way ANOVA indicated that ERK4 deficiency altered the effect of TAC on TGFβ 1 and collagen 1-α 1 transcript levels with each being higher in TAC-ERK4 -/- mice. Furthermore, MK5 immunoprecipitates from cardiac fibroblast lysates did not contain ERK4 immunoreactivity. Additional experiments revealed the presence of ERK4 immunoreactivity in myocytes but not fibroblasts. These results suggest 1) ERK4 may be involved in myocyte - fibroblast communication during myocardial remodeling and 2) in cardiac myocytes, ERK4 is part of a novel signaling cascade that does not involve MK5.

ERK3/MAPK6 controls IL-8 production and chemotaxis.

ERK3 is a ubiquitously expressed member of the atypical mitogen activated protein kinases (MAPKs) and the physiological significance of its short half-life remains unclear. By employing gastrointestinal 3D organoids, we detect that ERK3 protein levels steadily decrease during epithelial differentiation. ERK3 is not required for 3D growth of human gastric epithelium. However, ERK3 is stabilized and activated in tumorigenic cells, but deteriorates over time in primary cells in response to lipopolysaccharide (LPS). ERK3 is necessary for production of several cellular factors including interleukin-8 (IL-8), in both, normal and tumorigenic cells. Particularly, ERK3 is critical for AP-1 signaling through its interaction and regulation of c-Jun protein. The secretome of ERK3-deficient cells is defective in chemotaxis of neutrophils and monocytes both in vitro and in vivo. Further, knockdown of ERK3 reduces metastatic potential of invasive breast cancer cells. We unveil an ERK3-mediated regulation of IL-8 and epithelial secretome for chemotaxis.

Mitogen-activated protein kinase regulation of the phosphodiesterase RegA in early Dictyostelium development.

Mitogen-activated protein kinase (MAPK) regulation of cAMP-specific phosphodiesterase function has been demonstrated in mammalian cells and suspected to occur in other eukaryotes. Epistasis analysis in the soil amoeba Dictyostelium discoideum suggests the atypical MAPK Erk2 downregulates the function of the cAMP-specific phosphodiesterase RegA to regulate progression of the developmental life cycle. A putative MAPK docking motif located near a predicted MAPK phosphorylation site was characterized for contributions to RegA function and binding to Erk2 because a similar docking motif has been previously characterized in the mammalian PDE4D phosphodiesterase. The overexpression of RegA with alterations to this docking motif (RegAD-) restored RegA function to regA- cells based on developmental phenotypes, but low-level expression of RegAD- from the endogenous regA promoter failed to rescue wild-type morphogenesis. Co-immunoprecipitation analysis indicated that Erk2 associates with both RegA and RegAD-, suggesting the docking motif is not required for this association. Epistasis analysis between regA and the only other Dictyostelium MAPK, erk1, suggests Erk1 and RegA can function in different pathways but that some erk1- phenotypes may require cAMP signalling. These results imply that MAPK downregulation of RegA in Dictyostelium is accomplished through a different mechanism than MAPK regulation of cAMP-specific phosphodiesterases in mammalian cells and that the regulation in Dictyostelium does not require a proximal MAPK docking motif.

Abstract 4289: ERK3 negatively regulates IL-6/STAT3 signaling via SOCS3 in cancer cells

Abstract Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen activated protein kinase (MAPK) which is dysregulated in several cancers including lung cancer, breast cancer and melanoma. It has a single Ser-Glu-Gly (SEG) phospho-acceptor motif in its activation loop, instead of the Thr-Xaa-Tyr (TXY) motif conserved in the classical MAPKs. Moreover, compared to the conventional MAPKs, much less is known about the targets of ERK3 signaling. Hence, to elucidate the ERK3-regulated genes and pathways, we performed gene microarray analyses of both A549 lung cancer cells and A375 melanoma cells after treatment with either a siRNA specifically targeting ERK3 or a non-targeting control siRNA. Interestingly, IL-6 target genes were found to be highly upregulated upon ERK3 knockdown. Interleukin-6 (IL-6) is a pleiotropic cytokine which signals via the JAK/STAT3 pathway. Phosphorylation of STAT3 at Tyrosine 705 (Y705) by JAK activates STAT3 in response to IL-6 stimulation. IL-6 signaling is negatively regulated by a feedback inhibitor SOCS3 (Suppressor of cytokine signaling 3), which binds to and inhibits JAK kinase activity. Interestingly, we noted that ERK3 interacts with SOCS3 from a published Yeast-two-hybrid screening. We validated the interaction between ERK3 and SOCS3 by co-immunoprecipitation assay. In addition, we found that ERK3 downregulates the phosphorylation of STAT3 at Y705. In line with its negative regulation of oncogenic STAT3 activity, ERK3 inhibits melanoma cell growth, migration/invasion and colony formation in soft agar. Taken together, we have revealed a novel role for ERK3 in suppressing IL-6/STAT3 signaling via SOCS3 in cancer cells. Citation Format: Astha Shakya, Minyi Chen, Michael Markey, Weiwen Long. ERK3 negatively regulates IL-6/STAT3 signaling via SOCS3 in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4289.

Monitoring GPCR Induced Atypical MAPK p38 Signaling to Identify Key Regulators of Vascular Inflammation

Activation of the mitogen activated protein kinase (MAPK) p38 is a critical regulatory step in pro‐inflammatory signaling and a key target for therapeutic intervention in vascular diseases, including chronic inflammation and lung disease. Our recent studies defined a conserved ubiquitin driven pathway that regulates atypical p38 signaling by a subset of G protein‐coupled receptors (GPCR). Atypical p38 signaling is initiated by GPCR induced c‐Src phosphorylation of an autoinhibitory peptide in the E3 ubiquitin ligase NEDD4‐2. In turn, phospho‐activation releases the autoinhibitory peptide from the catalytic site of NEDD4‐2, a step which is critical for stress induced GPCR ubiquitination and amplification of atypical p38 signaling and inflammation in the vasculature. Despite the critical role of GPCR induced p38 signaling in the onset and progression of many pathophysiological responses, atypical p38 signaling is still relatively understudied. Here we report the development of Försters Resonance Energy Transfer (FRET) biosensors to map the spatiotemporal activation of p38 signaling by multiple GPCRs in vascular endothelial cells. These FRET sensors create an ideal platform for a small‐scale screening pipeline to identify molecular modulators of the atypical p38 signaling pathway. Our studies have already identified multiple regulatory proteins that block the induction and/or magnitude of GPCR induced p38 signaling, perturbing pro‐inflammatory signaling in the vasculature.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Germ Line Deletion Reveals a Nonessential Role of Atypical Mitogen-Activated Protein Kinase 6/Extracellular Signal-Regulated Kinase 3.

Mitogen-activated protein kinase 6/extracellular signal-regulated kinase 3 (MAPK6/ERK3) is an atypical member of the MAPKs. An essential role has been suggested by the perinatal lethal phenotype of ERK3 knockout mice carrying a lacZ insertion in exon 2 due to pulmonary dysfunction and by defects in function, activation, and positive selection of T cells. To study the role of ERK3 in vivo, we generated mice carrying a conditional Erk3 allele with exon 3 flanked by loxP sites. Loss of ERK3 protein was validated after deletion of Erk3 in the female germ line using zona pellucida 3 (Zp3)-cre and a clear reduction of the protein kinase MK5 is detected, providing the first evidence for the existence of the ERK3/MK5 signaling complex in vivo In contrast to the previously reported Erk3 knockout phenotype, these mice are viable and fertile and do not display pulmonary hypoplasia, acute respiratory failure, abnormal T-cell development, reduction of thymocyte numbers, or altered T-cell selection. Hence, ERK3 is dispensable for pulmonary and T-cell functions. The perinatal lethality and lung and T-cell defects of the previous ERK3 knockout mice are likely due to ERK3-unrelated effects of the inserted lacZ-neomycin resistance cassette. The knockout mouse of the closely related atypical MAPK ERK4/MAPK4 is also normal, suggesting redundant functions of both protein kinases.

Reevaluation of the Role of Extracellular Signal-Regulated Kinase 3 in Perinatal Survival and Postnatal Growth Using New Genetically Engineered Mouse Models.

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 (Mapk6lacZ ) showed that inactivation of ERK3 in Mapk6lacZ 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 (Mapk6KD ) or conditional (Mapk6Δ ) 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 Mapk6lacZ mice but not in Mapk6KD or Mapk6Δ 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 Mapk6lacZ mutant mice.