Activation loop phosphorylation of ERK3 is important for its kinase activity and ability to promote lung cancer cell invasiveness

Elkhadragy, Lobna; Alsaran, Hadel; Morel, Marion; Long, Weiwen

doi:10.1074/jbc.ra118.003699

Cited by 30 publications

(61 citation statements)

References 32 publications

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“…and ERK4 via an unusual motif located in their C-terminals called D domain and FRIEDE motif, respectively 49 . In the case of ERK3, phosphorylation at serine-189, but not its actual catalytic activity, is necessary for its binding to MK5 48,61,62 . We observed, co-immunoprecipitation of GAPDH AS 5′-ACT GTG GTC ATG AGC CCT TCC A-3′ S, sense; AS, anti-sense; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; MK, MAPK-activated protein kinase; qPCR, quantitative polymer chain reaction; ANP, atrial natriuretic peptide; β-MHC, β-myosin heavy chain.…”

Section: Discussionmentioning

confidence: 99%

Expression, subcellular localization, and phosphorylation of MK5 in adult cardiac ventricular fibroblasts

Sahadevan

Nawaito

Trépanier

et al. 2020

Preprint

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MAP kinase-activated protein kinase-5 (MK5) plays an important role in cardiac fibroblast function. Although p38 MAPK and atypical MAPKs and ERK3 and ERK4 have been identified as activators of MK5, the kinases that activate MK5 remain controversial. Here we examined the expression, subcellular distribution, and regulation of MK5 in cardiac ventricular myofibroblasts and myocytes. The copy numbers for MK5 and ERK4 mRNA were comparable in myocytes and myofibroblasts, whereas that of ERK3 was much higher in myofibroblasts. Interestingly, MK5 and ERK3 immunoreactivity was detected in myofibroblasts but not myocytes whereas ERK4 immunoreactivity was detected in myocytes: treating in myocytes with a proteasome inhibitor or hypertrophic agonists failed to rescue MK5 immunoreactivity. In myofibroblasts, MK5 and ERK3 immunoreactivity was predominantly nuclear and cytosolic, respectively. In serum-starved cardiac myofibroblasts, phosphothreonine-182 MK5 (pT182-MK5) immunoreactivity was predominantly nuclear but increased in intensity and relocated to the cytoplasm in response to serum, sorbitol, angiotensin II, TGFβ, or H2O2 and this was prevented by inhibition of p38α/β. Phos-tag SDS-PAGE revealed multiple slower migrating bands of MK5 immunoreactivity, indicating phosphorylation of MK5 at multiple sites. Phos-tag PAGE also revealed MK5 phosphorylation was increased with fibroblast activation and in hearts exposed to a chronic increase in afterload. MK5 and ERK3 co-immunoprecipitated and proximity ligation assays revealed ERK3 and MK5 in close proximity in myofibroblast cytoplasmic compartment. Furthermore, p38α/β inhibition decreased the abundance of MK5 immunoreactivity in ERK3 immunoprecipitates. Finally, deleting MK5 did not reduce the abundance of ERK3 immunoreactivity. These observations suggest that p38α and/or p38β are the primary mediators of T182-MK5 phosphorylation and hence MK5 activation in cardiac myofibroblasts.

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Section: Discussionmentioning

confidence: 99%

Expression, subcellular localization, and phosphorylation of MK5 in adult cardiac ventricular fibroblasts

Sahadevan

Nawaito

Trépanier

et al. 2020

Preprint

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“…Role of IL-8 in tumour progression has been well established (Chan et al, 2017;Long et al, 2016) and high levels of this chemokine are a poor prognostic factor in melanoma, breast, liver, lung and colon cancer (David et al, 2016;Ueda et al, 1994). Previous studies on ERK3 indicated its role in tumourigenesis, including regulation of migratory properties of MDA-MB231 breast cancer cells (Al-Mahdi et al, 2015;Elkhadragy et al, 2018). Similarly, IL-8 has been reported to enhance migration and thus metastasis of breast carcinoma cells MDA-MB231, which also carry a KRAS G13D mutation (Jayatilaka et al, 2017).…”

Section: Depletion Of Erk3 Reduces Metastatic Potential Of Breast Canmentioning

confidence: 99%

ERK3/MAPK6 controls IL-8 production and chemotaxis

Bogucka

Pompaiah

Marini

et al. 2020

eLife

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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.

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“…For transient transfection, A431 cells were transfected with empty vector control (EV) or ERK3 plasmids using Lipofectamine 3000 reagent (Thermo Fisher Scienti c, Carlsbad, CA, USA), and 48 hours after transfection cells were harvested and tested for protein expression. A431 cells were transduced with lentiviruses expressing an empty vector CDH or CDH-ERK3 as previously described [27].…”

Section: Cell Culture and Reagentsmentioning

confidence: 99%

“…Cells were lysed in the lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP-40, 1 mM complete protease inhibitors and 1 mM phosphatase inhibitor cocktail III (Sigma-Aldrich). Immunoblot analysis was carried as previously described [27]. The following primary antibodies were used in immunoblotting: Mouse monoclonal anti-pan-p63 (4A4), rabbit monoclonal anti-ERK3 (ab53277, 1:1000, Abcam), rabbit polyclonal anti-pRac1 S71 (sc-12924, 1:500, Santa Cruz), mouse monoclonal anti-Rac1(ab33186, 1:5000, Abcam) and mouse monoclonal anti-b-actin (A5316, 1:20,000, Sigma-Aldrich) antibodies were used to detect ΔNp63α, ERK3, pRac1, Rac1 and β-actin, respectively.…”

Section: Immunoblot Analysismentioning

confidence: 99%

ERK3 is transcriptionally upregulated by ∆Np63α and mediates the role of ∆Np63α in suppressing cell migration in Non-Melanoma Skin Cancers

Alshammari

Aljagthmi

Stacy

et al. 2020

Preprint

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Abstract Background: p63, 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. Methods: Fluorescent immunohistochemistry was performed to evaluate the expression levels of DNp63a 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 DNp63a and ERK3 expression levels (MFI). The regulation of ERK3 by DNp63a was studied by qRT-PCR, Western blot and luciferase assay. The effect of ERK3 regulation by DNp63a on cell migration was measured by performing trans-well migration assay. Results: The 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.Conclusions: ERK3 is positively regulated by ∆Np63α and mediates the role of ∆Np63α in suppressing cell migration in NMSC.

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Activation loop phosphorylation of ERK3 is important for its kinase activity and ability to promote lung cancer cell invasiveness

Cited by 30 publications

References 32 publications

Expression, subcellular localization, and phosphorylation of MK5 in adult cardiac ventricular fibroblasts

Expression, subcellular localization, and phosphorylation of MK5 in adult cardiac ventricular fibroblasts

ERK3/MAPK6 controls IL-8 production and chemotaxis

ERK3 is transcriptionally upregulated by ∆Np63α and mediates the role of ∆Np63α in suppressing cell migration in Non-Melanoma Skin Cancers

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