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An analysis of the phosphorylation and activation of extracellular-signal-regulated protein kinase 5 (ERK5) by mitogen-activated protein kinase kinase 5 (MKK5) in vitro.

ABSTRACT: MKK5 expressed as a glutathione S-transferase fusion protein in human embryonic kidney 293 cells activated full-length extracellular-signal-regulated protein kinase (ERK)5 (ERK5wt) as well as the isolated catalytic domain (ERK5cat) in vitro. Activation was accompanied by the phosphorylation of Thr(219) and Tyr(221), the former residue being phosphorylated preferentially. ERK5cat phosphorylated at Thr(219), but not Tyr(221), possessed 10% of the activity of the doubly phosphorylated protein towards myelin basic protein, whereas ERK5cat phosphorylated at Tyr(221) alone was much less active. Activated ERK5 phosphorylated itself at a number of residues, including Thr(28), Ser(421), Ser(433), Ser(496), Ser(731) and Thr(733). ERK5 phosphorylated at Thr(219), but not Tyr(221), phosphorylated itself at a similar rate to ERK5 phosphorylated at both Thr(219) and Tyr(221). Activated ERK5 also phosphorylated mitogen-activated protein kinase kinase 5 (MKK5) extensively at Ser(129), Ser(137), Ser(142) and Ser(149), which are located within the region in MKK5 that is thought to interact with ERK5.

SUBMITTER: Mody N

PROVIDER: S-EPMC1223423 | biostudies-other | 2003 Jun

REPOSITORIES: biostudies-other

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Activation of mitogen-activated protein kinase (mitogen-activated protein kinase/extracellular signal-regulated kinase) cascade by aldosterone.

Project description:Aldosterone in some tissues increases expression of the mRNA encoding the small monomeric G protein Ki-RasA. Renal A6 epithelial cells were used to determine whether induction of Ki-ras leads to concomitant increases in the total as well as active levels of Ki-RasA and whether this then leads to subsequent activation of its effector mitogen-activated protein kinase (MAPK/extracellular signal-regulated kinase) cascade. The molecular basis and cellular consequences of this action were specifically investigated. We identified the intron 1-exon 1 region (rasI/E1) of the mouse Ki-ras gene as sufficient to reconstitute aldosterone responsiveness to a heterologous promotor. Aldosterone increased reporter gene activity containing rasI/E1 threefold. Aldosterone increased the absolute and GTP-bound levels of Ki-RasA by a similar extent, suggesting that activation resulted from mass action and not effects on GTP binding/hydrolysis rates. Aldosterone significantly increased Ki-RasA and MAPK activity as early as 15 min with activation peaking by 2 h and waning after 4 h. Inhibitors of transcription, translation, and a glucocorticoid receptor antagonist attenuated MAPK signaling. Similarly, rasI/E1-driven luciferase expression was sensitive to glucocorticoid receptor blockade. Overexpression of dominant-negative RasN17, addition of antisense Ki-rasA and inhibition of mitogen-activated protein kinase kinase also attenuated steroid-dependent increases in MAPK signaling. Thus, activation of MAPK by aldosterone is dependent, in part, on a genomic mechanism involving induction of Ki-ras transcription and subsequent activation of its downstream effectors. This genomic mechanism has a distinct time course from activation by traditional mitogens, such as serum, which affect the GTP-binding state and not absolute levels of Ras. The result of such a genomic mechanism is that peak activation of the MAPK cascade by adrenal corticosteroids is delayed but prolonged.

| S-EPMC124141 | biostudies-literature

A novel interplay between Epac/Rap1 and mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) regulates thrombospondin to control angiogenesis.

Project description:Tumors depend upon angiogenesis for growth and metastasis. It is therefore critical to understand the inhibitory signaling mechanisms in endothelial cells that control angiogenesis. Epac is a cyclic adenosine 5'-monophosphate-activated guanine nucleotide exchange factor for Rap1. In this study, we show that activation of Epac or Rap1 leads to potent inhibition of angiogenesis in vivo. Epac/Rap1 activation down-regulates inhibitor of differentiation 1 (Id1), which negatively regulates thrombospondin-1 (TSP1), an inhibitor of angiogenesis. Consistent with this mechanism, activation of Epac/Rap 1 induces expression of TSP1; conversely, depletion of Epac reduces TSP1 levels in endothelial cells. Blockade of TSP1 binding to its receptor, CD36, rescues inhibition of chemotaxis or angiogenesis by activated Epac/Rap1. Mitogen-activated protein kinase kinase 5, a downstream mediator of vascular endothelial growth factor, antagonizes the effects of Epac/Rap1 by inducing Id1 and suppressing TSP1 expression. Finally, TSP1 is also secreted by fibroblasts in response to Epac/Rap1 activation. These results identify Epac and Rap1 as inhibitory regulators of the angiogenic process, implicate Id1 and TSP1 as downstream mediators of Epac/Rap1, and highlight a novel interplay between pro- and antiangiogenic signaling cascades involving multiple cell types within the angiogenic microenvironment.

| S-EPMC2777131 | biostudies-literature

Reciprocal regulation of extracellular signal regulated kinase 1/2 and mitogen activated protein kinase phosphatase-3.

Project description:Mitogen activated protein kinase phosphatase-3 (MKP-3) is a putative tumor suppressor. When transiently overexpressed, MKP-3 dephosphorylates and inactivates extracellular signal regulated kinase (ERK) 1/2. Little is known about the roles of endogenous MKP-3, however. We previously showed that MKP-3 is upregulated in cell lines that express oncogenic Ras. Here we tested the roles of endogenous MKP-3 in modulating ERK1/2 under conditions of chronic stimulation of the Ras/Raf/MEK1/2/ERK1/2 pathway by expression of oncogenic Ras. We used two cell lines: H-ras MCF10A, breast epithelial cells engineered to express H-Ras, and DLD-1, colon cancer cells that express endogenous Ki-Ras. First, we found that MKP-3 acts in a negative feedback loop to suppress basal ERK1/2 when oncogenic Ras stimulates the Ras/Raf/MEK1/2/ERK1/2 cascade. ERK1/2 was required to maintain elevated MKP-3, indicative of a negative feedback loop. Accordingly, knockdown of MKP-3, via siRNA, increased ERK1/2 phosphorylation. Second, by using siRNA, we found that MKP-3 helps establish the sensitivity of ERK1/2 to extracellular activators by limiting the duration of ERK1/2 phosphorylation. Third, we found that the regulation of ERK1/2 by MKP-3 is countered by the complex regulation of MKP-3 by ERK1/2. Potent ERK1/2 activators stimulated the loss of MKP-3 within 30 min due to an ERK1/2-dependent decrease in MKP-3 protein stability. MKP-3 levels recovered within 120 min due to ERK1/2-dependent resynthesis. Preventing MKP-3 resynthesis, via siRNA, prolonged ERK1/2 phosphorylation. Altogether, these results suggest that under the pressure of oncogenic Ras expression, MKP-3 reins in ERK1/2 by serving in ERK1/2-dependent negative feedback pathways.

| S-EPMC2581931 | biostudies-literature

Mitogen-activated protein kinase extracellular signal-regulated kinase 2 phosphorylates and promotes Pin1 protein-dependent promyelocytic leukemia protein turnover.

Project description:The promyelocytic leukemia (PML) protein is a tumor suppressor that has an important role in several cellular processes, including apoptosis, viral infection, DNA damage repair, cell cycle regulation, and senescence. PML is an essential component of sub-nuclear structures called PML nuclear bodies (NBs). Our laboratory has previously demonstrated that the peptidyl-prolyl cis-trans isomerase, Pin1, binds and targets PML for degradation in a phosphorylation-dependent manner. To further elucidate the mechanisms underlying Pin1-mediated PML degradation, we aimed to identify one or more factors that promote PML phosphorylation. Here we show that treatment with U0126, an inhibitor of the ERK2 upstream kinases MEK1/2, leads to an increase in PML protein accumulation and an inhibition of the interaction between Pin1 and PML in MDA-MB-231 breast cancer cells. Consistent with this observation, phosphorylated ERK2 partially co-localized with PML NBs. Although U0126 up-regulated exogenous wild-type PML levels, it did not have an effect on the steady-state level of a mutant form of PML that is defective in binding Pin1. In addition, exogenous wild-type, but not Pin1 binding-defective PML protein expression levels were decreased by overexpression of ERK2. In contrast, knockdown of ERK2 by siRNA resulted in an increase in PML protein levels and an increase in the formation of PML NBs. Using phospho-specific antibodies, we identified Ser-403 and Ser-505 as the ERK2 targets that promote Pin1-mediated PML degradation. Finally, we demonstrated that EGF induced activation of ERK and interaction between PML and phosphorylated ERK resulting in a decrease in PML protein levels. Taken together, our results support a model in which Pin1 promotes PML degradation in an ERK2-dependent manner.

| S-EPMC3248013 | biostudies-literature

In vitro and in vivo radiosensitization with AZD6244 (ARRY-142886), an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 kinase.

Project description:PURPOSE:The mitogen-activated protein (MAP) kinase pathway is important for cell proliferation, survival, and differentiation, and is frequently up-regulated in cancers. The MAP kinase pathway is also activated after exposure to ionizing radiation. We investigated the effects of AZD6244 (ARRY-142886), an inhibitor of MAP kinase/extracellular signal-regulated kinase 1/2, on radiation response. EXPERIMENTAL DESIGN:The effects of AZD6244 on the in vitro radiosensitivity of human cancer cell lines (A549, MiaPaCa2, and DU145) were evaluated using clonogenic assays. DNA damage repair was evaluated using gammaH2AX, and mitotic catastrophe was measured using nuclear fragmentation. Cell cycle effects were measured with flow cytometry. Growth delay was used to evaluate the effects of AZD6244 on in vivo tumor radiosensitivity. RESULTS:Exposure of each cell line to AZD6244 before irradiation resulted in an increase in radiosensitivity with dose enhancement factors at a surviving fraction of 0.1, ranging from 1.16 to 2.0. No effects of AZD6244 on radiation-induced apoptosis or persistence of gammaH2AX foci after irradiation were detected. Cells treated with AZD6244 had an increased mitotic index and decreased Chk1 phosphorylation at 1 and 2 hours after irradiation. Mitotic catastrophe was increased in cells receiving AZD6244 and irradiation compared with the single treatments. In vivo studies revealed that AZD6244 administration to mice bearing A549 tumor xenografts resulted in a greater than additive increase in radiation-induced tumor growth delay (dose enhancement factor of 3.38). CONCLUSIONS:These results indicate that AZD6244 can enhance tumor cell radiosensitivity in vitro and in vivo and suggest that this effect involves an increase in mitotic catastrophe.

| S-EPMC2798161 | biostudies-literature

Brain-specific deletion of extracellular signal-regulated kinase 2 mitogen-activated protein kinase leads to aberrant cortical collagen deposition.

Project description:The mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1 and 2 are essential intracellular mediators of numerous transmembrane signals. To investigate neural-specific functions of ERK2 in the brain, we used a Cre/lox strategy using Nestin:Cre to drive recombination in neural precursor cells. Nestin:Cre;ERK2(fl/fl) conditional knockout (cKO) mice have architecturally normal brains and no gross behavioral deficits. However, all cKO mice developed early-onset (postnatal day 35 to 40) frontal cortical astrogliosis, without evidence of neuronal degeneration. Frontoparietal cortical gray matter, but not underlying white matter, was found to contain abundant pericapillary and parenchymal reticulin fibrils, which were shown by immunohistochemistry to contain fibrillar collagens, including type I collagen. ERK1 general KO mice showed neither fibrils nor astrogliosis, indicating a specific role for ERK2 in the regulation of brain collagen. Collagen fibrils were also observed to a lesser extent in GFAP:Cre;ERK2(fl/fl) mice but not in CamKII-Cre;ERK2(fl/fl) mice (pyramidal neuron specific), consistent with a possible astroglial origin. Primary astroglial cultures from cKO mice expressed elevated fibrillar collagen levels, providing further evidence that the phenotype may be cell autonomous for astroglia. Unlike most other tissues, brain and spinal cord parenchyma do not normally contain fibrillar collagens, except in disease states. Determining mechanisms of ERK2-mediated collagen regulation may enable targeted suppression of glial scar formation in diverse neurological disorders.

| S-EPMC2789603 | biostudies-literature

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

Project description: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.

| S-EPMC6399663 | biostudies-literature

Integrins regulate microtubule nucleating activity of centrosome through mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling.

Project description:Microtubule nucleation is an essential step in the formation of the microtubule cytoskeleton. We recently showed that androgen and Src promote microtubule nucleation and ?-tubulin accumulation at the centrosome. Here, we explore the mechanisms by which androgen and Src regulate these processes and ask whether integrins play a role. We perturb integrin function by a tyrosine-to-alanine substitution in membrane-proximal NPIY motif in the integrin ?1 tail and show that this mutant substantially decreases microtubule nucleation and ?-tubulin accumulation at the centrosome. Because androgen stimulation promotes the interaction of the androgen receptor with Src, resulting in PI3K/AKT and MEK/ERK signaling, we asked whether these pathways are inhibited by the mutant integrin and whether they regulate microtubule nucleation. Our results indicate that the formation of the androgen receptor-Src complex and the activation of downstream pathways are significantly suppressed when cells are adhered by the mutant integrin. Inhibitor studies indicate that microtubule nucleation requires MEK/ERK but not PI3K/AKT signaling. Importantly, the expression of activated RAF-1 is sufficient to rescue microtubule nucleation inhibited by the mutant integrin by promoting the centrosomal accumulation of ?-tubulin. Our data define a novel paradigm of integrin signaling, where integrins regulate microtubule nucleation by promoting the formation of androgen receptor-Src signaling complexes to activate the MEK/ERK signaling pathway.

| S-EPMC3268412 | biostudies-other

Novel phosphorylation-dependent ubiquitination of tristetraprolin by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) and tumor necrosis factor receptor-associated factor 2 (TRAF2).

Project description:Acute versus chronic inflammation is controlled by the accurate activation and regulation of interdependent signaling cascades. TNF-receptor 1 engagement concomitantly activates NF-κB and JNK signaling. The correctly timed activation of these pathways is the key to account for the balance between NF-κB-mediated cell survival and cell death, the latter fostered by prolonged JNK activation. Tristetraprolin (TTP), initially described as an mRNA destabilizing protein, acts as negative feedback regulator of the inflammatory response: it destabilizes cytokine-mRNAs but also acts as an NF-κB inhibitor by interfering with the p65/RelA nuclear import pathway. Our biochemical studies provide evidence that TTP contributes to the NF-κB/JNK balance. We find that the MAP 3-kinase MEKK1 acts as a novel TTP kinase that, together with the TNF receptor-associated factor 2 (TRAF2), constitutes not only a main determinate of the NF-κB-JNK cross-talk but also facilitates "TTP hypermodification": MEKK1 triggers TTP phosphorylation as prerequisite for its Lys-63-linked, TRAF2-mediated ubiquitination. Consequently, TTP no longer affects NF-κB activity but promotes the activation of JNK. Based on our data, we suggest a model where upon TNFα induction, TTP transits a hypo- to hypermodified state, thereby contributing to the molecular regulation of NF-κB versus JNK signaling cascades.

| S-EPMC3207473 | biostudies-literature

Phorbol ester-induced activation of mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase and extracellular-signal-regulated protein kinase decreases glucose-6-phosphatase gene expression.

Project description:Glucose-6-phosphatase (G6Pase) plays a central role in blood glucose homoeostasis, and insulin suppresses G6Pase gene expression by the activation of phosphoinositide 3-kinase (PI 3-kinase). Here, we show that the phorbol ester PMA decreases both basal and dexamethasone/cAMP-induced expression of a luciferase gene under the control of the G6Pase promoter in transiently transfected H4IIE hepatoma cells. This regulation was suppressed by the inhibitors of the mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK), PD98059 and U0126, but not by the inhibitor of PI 3-kinase, LY294002. The co-expression of a constitutively active mutant of MEK mimicked the regulation of G6Pase promoter activity by PMA. The effect of PMA on both basal and induced G6Pase gene transcription was impaired by the overexpression of a dominant negative MEK construct, as well as by the expression of mitogen-activated protein kinase phosphatase-1. The mutation of the forkhead-binding sites within the insulin-response unit of the G6Pase promoter, which decreases the effect of insulin on G6Pase gene expression, did not alter the regulation of gene expression by PMA. The data show that PMA decreases G6Pase gene expression by the activation of MEK and extracellular-signal regulated protein kinase. With that, PMA mimics the effect of insulin on G6Pase gene expression by a different signalling pathway.

| S-EPMC1222018 | biostudies-other

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