The N-terminal ERK-binding site of MEK1 is required for efficient feedback phosphorylation by ERK2 in vitro and ERK activation in vivo

An ERK2-binding site at the N terminus of MEK1 was reported to mediate their stable association. We examined the importance of this binding site in the feedback phosphorylation of MEK1 on Thr(292) and Thr(386) by ERK2, the phosphorylation and activation of ERK2 by MEK1, and the interaction of MEK1 with ERK2 and Raf-1. Deletion of the binding site from MEK1 reduced its phosphorylation by ERK2, but had no effect on its phosphorylation by p21-activated protein kinase-1 (PAK1). A MEK1 N-terminal peptide containing the binding site inhibited MEK1 phosphorylation by ERK2. However, it did not affect MEK1 phosphorylation by p21-activated protein kinase or myelin basic protein phosphorylation by ERK2. Deletion of the N-terminal ERK-binding domain of MEK1 also reduced its ability to phosphorylate ERK2 in vitro, to co-immunoprecipitate with ERK2, and to stimulate ERK2 activation in transfected cells, but it did not alter the association with endogenous Raf-1. Using ERK2-p38 chimeras and an ERK2 deletion mutant, a MEK1-binding site of ERK2 was localized to its N terminus.     

Insulin reverses growth hormone-induced homologous desensitization

Growth hormone (GH) is secreted in a pulsatile pattern to promote body growth and metabolism. GH exerts its function by activating several signaling pathways, including JAK2/STAT and MEK/ERK. ERK1/2 activation by GH plays important roles in gene expression, cell proliferation, and growth. We previously reported that in rat H4IIE hepatoma cells after an initial GH exposure, a second GH exposure induces STAT5 phosphorylation but not ERK1/2 phosphorylation (Ji, S., Frank, S. J., and Messina, J. L. (2002) J. Biol. Chem. 277, 28384-28393). In this study the mechanisms underlying GH-induced homologous desensitization were investigated. A second GH exposure activated the signaling intermediates upstream of MEK/ERK, including JAK2, Ras, and Raf-1. This correlated with recovery of GH receptor levels, but was insufficient for GH-induced phosphorylation of MEK1/2 and ERK1/2. Insulin restored the ability of a second GH exposure to induce phosphorylation of MEK1/2 and ERK1/2 without altering GH receptor levels or GH-induced phosphorylation/activation of JAK2 and Raf-1. GH and insulin synergized in promoting cell proliferation. Further investigation suggested that insulin increased the amount of MEK bound to KSR (kinase suppressor of Ras) and restored GH-induced tyrosine phosphorylation of KSR. Previous GH exposure also induced desensitization of STAT1 and STAT3 phosphorylation, but this desensitization was not reversed by insulin. Thus, insulin-regulated resensitization of GH signaling may be necessary to reset the complete response to GH after a normal, physiologic pulse of GH.     

IL-2 activation of a PI3K-dependent STAT3 serine phosphorylation pathway in primary human T cells

Interleukin-2 (IL-2) is the major growth factor of activated T lymphocytes. By inducing cell cycle progression and protection from apoptosis in these cells, IL-2 is involved in the successful execution of an immune response. Upon binding its receptor, IL-2 activates a variety of signal transduction pathways, including the Ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) and Janus kinase (JAK)/STAT cascades. In addition, activation of phosphatidylinositol 3-kinase (PI3K) and several of its downstream targets has also been shown. However, the coupling of STAT3 serine phosphorylation to PI3K in response to IL-2 has yet to be shown in either T cell lines or primary human T cells. This report shows that the PI3K inhibitors LY294002 and wortmannin block activation of MEK and ERK by IL-2 in primary human T cells. Moreover, these inhibitors significantly reduce IL-2-triggered STAT3 serine phosphorylation without affecting STAT5 serine phosphorylation. Analysis of the effects of these inhibitors on cell cycle progression and apoptosis strongly suggests that PI3K-mediated events, which includes STAT3 activation, are involved in IL-2-mediated cell proliferation but not cell survival. Finally, results presented illustrate that in primary human T cells, activation of Akt is insufficient for IL-2-induced anti-apoptosis. Thus, these results demonstrate that IL-2 stimulates PI3K-dependent events that correlate with cell cycle progression, but not anti-apoptosis, in activated primary human T cells.     

Regulation of ERK Kinase by MEK1 Kinase Inhibition in the Brain

Metabotropic (slow) and ionotropic (fast) neurotransmission are integrated by intracellular signal transduction mechanisms involving protein phosphorylation/dephosphorylation to achieve experience-dependent alterations in brain circuitry. ERK is an important effector of both slow and fast forms of neurotransmission and has been implicated in normal brain function and CNS diseases. Here we characterize phosphorylation of the ERK-activating protein kinase MEK1 by Cdk5, ERK, and Cdk1 in vitro in intact mouse brain tissue and in the context of an animal behavioral paradigm of stress. Cdk5 only phosphorylates Thr-292, whereas ERK and Cdk1 phosphorylate both Thr-292 and Thr-286 MEK1. These sites interact in a kinase-specific manner and inhibit the ability of MEK1 to activate ERK. Thr-292 and Thr-286 MEK1 are phosphorylated in most mouse brain regions to stoichiometries of ∼5% or less. Phosphorylation of Thr-292 MEK1 is regulated by cAMP-dependent signaling in mouse striatum in a manner consistent with negative feedback inhibition in response to ERK activation. Protein phosphatase 1 and 2A contribute to the maintenance of the basal phosphorylation state of both Thr-292 and Thr-286 MEK1 and that of ERK. Activation of the NMDA class of ionotropic glutamate receptors reduces inhibitory MEK1 phosphorylation, whereas forced swim, a paradigm of acute stress, attenuates Thr-292 MEK1 phosphorylation. Together, the data indicate that these inhibitory MEK1 sites phosphorylated by Cdk5 and ERK1 serve as mechanistic points of convergence for the regulation of ERK signaling by both slow and fast neurotransmission.     

白藜芦醇通过下调MEK/ERK/c-Jun信号通路抑制H2O2诱导的肺癌细胞增殖

目的:探讨白藜芦醇(Res)是否通过下调ERK激酶/胞外信号调节激酶/原癌基因(MEK/ERK/c-Jun)信号通路抑制小剂量过氧化氢(H2O2)诱导肺癌细胞增殖。方法:采用MTS实验检测小剂量20μM H2O2以及分别加入MEK阻断剂U0126和Res后H2O2对肺癌细胞NCI-H1395增殖的影响,采用Western Blot检测H2O2对ERK1/2和Akt蛋白磷酸化水平以及加入Res后H2O2对MEK、ERK1/2和c-Jun蛋白磷酸化水平的影响。结果:小剂量H2O2对肺癌细胞NCI-H1395具有促增殖作用,H2O2通过活化ERK1/2和Akt蛋白的磷酸化水平促进肺癌细胞NCI-H1395增殖,加入MEK阻断剂U0126后H2O2对肺癌细胞NCI-H1395增殖作用降低(P<0.05)。Res可抑制H2O2诱导的肺癌细胞NCI-H1395增殖,加入Res后,H2O2引起的MEK、ERK1/2和c-Jun蛋白磷酸化水平均降低(P<0.05)。结论:小剂量H2O2对肺癌细胞NCI-H1395具有促增殖作用,Res通过抑制MEK/ERK/c-Jun信号通路来抑制H2O2对肺癌细胞NCI-H1395的促增殖作用,其具体机制还需进一步研究。     

Role of PKC and MAPK in cytosolic PLA2 phosphorylation and arachadonic acid release in primary murine astrocytes

Although Group IV cytosolic phospholipase A2 (cPLA2) in astrocytes has been implicated in a number of neurodegenerative diseases, mechanisms leading to its activation and release of arachidonic acid (AA) have not been clearly elucidated. In primary murine astrocytes, phorbol myristate acetate (PMA) and ATP stimulated phosphorylation of ERK1/2 and cPLA2 as well as evoked AA release. However, complete inhibition of phospho-ERK by U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), did not completely inhibit PMA-stimulated cPLA2 and AA release. Epidermal growth factor (EGF) also stimulated phosphorylation of ERK1/2 and cPLA2[largely through a protein kinase C (PKC)-independent pathway], but EGF did not evoke AA release. These results suggest that phosphorylation of cPLA2 due to phospho-ERK is not sufficient to evoke AA release. However, complete inhibition of ATP-induced cPLA2 phosphorylation and AA release was observed when astrocytes were treated with GF109203x, a general PKC inhibitor, together with U0126, indicating the important role for both PKC and ERK in mediating the ATP-induced AA response. There is evidence that PMA and ATP stimulated AA release through different PKC isoforms in astrocytes. In agreement with the sensitivity of PMA-induced responses to PKC down-regulation, prolonged treatment with PMA resulted in down-regulation of PKCalpha and epsilon in these cells. Furthermore, PMA but not ATP stimulated rapid translocation of PKCalpha from cytosol to membranes. Together, our results provided evidence for an important role of PKC in mediating cPLA2 phosphorylation and AA release in astrocytes through both ERK1/2-dependent and ERK1/2-independent pathways.     

Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro.

Raf-1 is a serine/threonine kinase which is essential in cell growth and differentiation. Tyrosine kinase oncogenes and receptors and p21ras can activate Raf-1, and recent studies have suggested that Raf-1 functions upstream of MEK (MAP/ERK kinase), which phosphorylates and activates ERK. To determine whether or not Raf-1 directly activates MEK, we developed an in vitro assay with purified recombinant proteins. Epitope-tagged versions of Raf-1 and MEK and kinase-inactive mutants of each protein were expressed in Sf9 cells, and ERK1 was purified as a glutathione S-transferase fusion protein from bacteria. Raf-1 purified from Sf9 cells which had been coinfected with v-src or v-ras was able to phosphorylate kinase-active and kinase-inactive MEK. A kinase-inactive version of Raf-1 purified from cells that had been coinfected with v-src or v-ras was not able to phosphorylate MEK. Raf-1 phosphorylation of MEK activated it, as judged by its ability to stimulate the phosphorylation of myelin basic protein by glutathione S-transferase-ERK1. We conclude that MEK is a direct substrate of Raf-1 and that the activation of MEK by Raf-1 is due to phosphorylation by Raf-1, which is sufficient for MEK activation. We also tested the ability of protein kinase C to activate Raf-1 and found that, although protein kinase C phosphorylation of Raf-1 was able to stimulate its autokinase activity, it did not stimulate its ability to phosphorylate MEK.     

Phosphorylation of threonine 290 in the activation loop of Tpl2/Cot is necessary but not sufficient for kinase activity

Cot/Tpl2/MAP3K8 is a serine/threonine kinase known to activate the ERK, p38, and JNK kinase pathways. Studies of Tpl2 knock-out mice reveal a clear defect in tumor necrosis factor-alpha production, although very little detail is known about its regulation and the signaling events involved. In the present study we demonstrated that phosphorylation of Cot was required for its maximal activity as phosphatase treatment of Cot decreased its kinase activity. The Cot sequence contains a conserved threonine at position 290 in the activation loop of the kinase domain. We found that mutation of this residue to alanine eliminated its ability to activate MEK/ERK and NF-kappaB pathways, whereas a phosphomimetic mutation to aspartic acid could rescue the ability to activate MEK. Thr-290 was also required for robust autophosphorylation of Cot. Antibody generated to phospho-Thr-290-Cot recognized both wild-type and kinase-dead Cot, suggesting that phosphorylation of Thr-290 did not occur through autophosphorylation but via another kinase. We showed that Cot was constitutively phosphorylated at Thr-290 in transfected human embryonic kidney 293T cells as well as human monocytes as this residue was phosphorylated in unstimulated and lipopolysaccharide-stimulated cells to the same degree. Treatment with herbimycin A inhibited Cot activity in the MEK/ERK pathway but did not inhibit phosphorylation at Thr-290. Together these results showed that phosphorylation of Cot at Thr-290 is necessary but not sufficient for full kinase activity in the MEK/ERK pathway.     

A monoclonal anti-IgE antibody against an epitope (amino acids 367-376) in the CH3 domain inhibits IgE binding to the low affinity IgE receptor (CD23)

We have produced three different mAb specific for human IgE-Fc. Their binding pattern to either heat-denatured IgE or a family of overlapping IgE-derived recombinant peptides and their ability to affect interaction of IgE with its low affinity receptor Fc epsilon R2/CD23 demonstrate that they recognize distinct epitopes on the IgE molecule. All three mAb were able to induce basophil degranulation as measured by the induction of histamine release. mAb 173 recognizes a thermolabile epitope in the CH4 domain. It does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 272 recognizes a thermostable epitope that maps to a sequence of 36 amino acids (AA) spanning part of the CH2 and CH3 domain and it does not affect the binding of IgE to Fc epsilon R2/CD23. mAb 27 recognizes a thermolabile epitope located on a 10 AA stretch (AA 367-376) in the CH3 domain. This area contains one N-linked oligosaccharide (Asn-371), but the antibody is not directed against carbohydrate because it binds to Escherichia coli-derived IgE peptides. mAb 27 inhibits the binding of IgE to Fc epsilon R2/CD23 but is still capable of reacting with IgE already bound to Fc epsilon R2/CD23. These data suggest that upon binding to Fc epsilon R2/CD23, the IgE molecule engages one of two equivalent-binding sites close to the glycosylated area of the CH3 domain.     

Human ERK1 induces filamentous growth and cell wall remodeling pathways in Saccharomyces cerevisiae

Expression of an activated extracellular signal-regulated kinase 1 (ERK1) construct in yeast cells was used to examine the conservation of function among mitogen-activated protein (MAP) kinases. Sequence alignment of the human MAP kinase ERK1 with all Saccharomyces cerevisiae kinases reveals a particularly strong kinship with Kss1p (invasive growth promoting MAP kinase), Fus3p (pheromone response MAP/ERK kinase), and Mpk1p (cell wall remodeling MAP kinase). A fusion protein of constitutively active human MAP/ERK kinase 1 (MEK) and human ERK1 was introduced under regulated expression into yeast cells. The fusion protein (MEK/ERK) induced a filamentation response element promoter and led to a growth retardation effect concomitant with a morphological change resulting in elongated cells, bipolar budding, and multicell chains. Induction of filamentous growth was also observed for diploid cells following MEK/ERK expression in liquid culture. Neither haploids nor diploids, however, showed marked penetration of agar medium. These effects could be triggered by either moderate MEK/ERK expression at 37 degrees C or by high level MEK/ERK expression at 30 degrees C. The combination of high level MEK/ERK expression and 37 degrees C resulted in cell death. The deleterious effects of MEK/ERK expression and high temperature were significantly mitigated by 1 m sorbitol, which also enhanced the filamentous phenotype. MEK/ERK was able to constitutively activate a cell wall maintenance reporter gene, suggesting misregulation of this pathway. In contrast, MEK/ERK effectively blocked expression from a pheromone-responsive element promoter and inhibited mating. These results are consistent with MEK/ERK promoting filamentous growth and altering the cell wall through its ability to partially mimic Kss1p and stimulate a pathway normally controlled by Mpk1p, while appearing to inhibit the normal functioning of the structurally related yeast MAP kinase Fus3p.     

Phosphatidylinositol 3-kinase-dependent mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 and NF-kappa B signaling pathways are required for B cell antigen receptor-mediated cyclin D2 induction in mature B cells

Phosphatidylinositol 3-kinase (PI-3K) has been linked to promitogenic responses in splenic B cells following B cell Ag receptor (BCR) cross-linking; however identification of the signaling intermediates that link PI-3K activity to the cell cycle remains incomplete. We show that cyclin D2 induction is blocked by the PI-3K inhibitors wortmannin and LY294002, which coincides with impaired BCR-mediated mitogen-activated protein/extracellular signal-related kinase kinase (MEK)1/2 and p42/44ERK phosphorylation on activation residues. Cyclin D2 induction is virtually absent in B lymphocytes from mice deficient in the class I(A) PI-3K p85alpha regulatory subunit. In contrast to studies with PI-3K inhibitors, which inhibit all classes of PI-3Ks, the p85alpha regulatory subunit is not required for BCR-induced MEK1/2 and p42/44ERK phosphorylation, suggesting the contribution of another PI-3K family members in MEK1/2 and p42/44ERK activation. However, p85alpha(-/-) splenic B cells are defective in BCR-induced IkappaB kinase beta and IkappaBalpha phosphorylation. We demonstrate that NF-kappaB signaling is required for cyclin D2 induction via the BCR in normal B cells, implicating a possible link with the defective IkappaB kinase beta and IkappaBalpha phosphorylation in p85alpha(-/-) splenic B cells and their ability to induce cyclin D2. These results indicate that MEK1/2-p42/44ERK and NF-kappaB pathways link PI-3K activity to Ag receptor-mediated cyclin D2 induction in splenic B cells.     

Cutting edge: extracellular signal-regulated kinase activates syk: a new potential feedback regulation of Fc epsilon receptor signaling.

The protein tyrosine kinase Syk is an essential element in several cascades coupling Ag receptors to cell responses. Syk and the mitogen-activated protein kinase extracellular signal-regulated kinase 1 (ERK1) were found to form a tight complex in both resting and Ag-stimulated rat mucosal-type mast cells (rat basophilic leukemia 2H3 cell line RBL-2H3). A direct serine phosphorylation and activation of Syk by ERK was observed in in vitro experiments. Moreover the mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) kinase (MEK) inhibitors markedly decreased the Ag-induced phosphorylation of the tyrosyl residues of Syk and its activation as well as suppressed the degranulation of the cells. These results suggest a positive feedback regulation of Syk by ERK in the cascade coupling the type 1 Fc epsilon receptor to the secretory response of mast cells; hence, the existence of a novel type of cross-talk between protein serine/threonine kinases and protein tyrosine kinases is suggested.     

Co-aggregation of FcgammaRII with FcepsilonRI on human mast cells inhibits antigen-induced secretion and involves SHIP-Grb2-Dok complexes

Signaling through the high affinity IgE receptor FcepsilonRI on human basophils and rodent mast cells is decreased by co-aggregating these receptors to the low affinity IgG receptor FcgammaRII. We used a recently described fusion protein, GE2, which is composed of key portions of the human gamma1 and the human epsilon heavy chains, to dissect the mechanisms that lead to human mast cell and basophil inhibition through co-aggregation of FcgammaRII and FcepsilonRI. Unstimulated human mast cells derived from umbilical cord blood express the immunoreceptor tyrosine-based inhibitory motif-containing receptor FcgammaRII but not FcgammaRI or FcgammaRIII. Interaction of the mast cells with GE2 alone did not cause degranulation. Co-aggregating FcepsilonRI and FcgammaRII with GE2 1) significantly inhibited IgE-mediated histamine release, cytokine production, and Ca(2+) mobilization, 2) reduced the antigen-induced morphological changes associated with mast cell degranulation, 3) reduced the tyrosine phosphorylation of several cellular substrates, and 4) increased the tyrosine phosphorylation of the adapter protein downstream of kinase 1 (p62(dok); Dok), growth factor receptor-bound protein 2 (Grb2), and SH2 domain containing inositol 5-phosphatase (SHIP). Tyrosine phosphorylation of Dok was associated with increased binding to Grb2. Surprisingly, in non-stimulated cells, there were complexes of phosphorylated SHIP-Grb2-Dok that were lost upon IgE receptor activation but retained under conditions of Fcepsilon-Fcgamma co-aggregation. Finally, studies using mast cells from Dok-1 knock-out mice showed that IgE alone triggers degranulation supporting an inhibitory role for Dok degranulation. Our results demonstrate how human FcepsilonRI-mediated responses can be inhibited by co-aggregation with FcgammaRIIB and implicate Dok, SHIP, and Grb2 as key intermediates in regulating antigen-induced mediator release.     

Peroxynitrite activates mitogen-activated protein kinase (MAPK) via a MEK-independent pathway: a role for protein kinase C.

In this study we show that phosphorylation of extracellular signal-regulated kinase (ERK1/2; also known as p44/42MAPK) following peroxynitrite (ONOO(-)) exposure occurs via a MAPK kinase (MEK)-independent but PKC-dependent pathway in rat-1 fibroblasts. ONOO(-)-mediated ERK1/2 phosphorylation was not blocked by MEK inhibitors PD98059 and U0126. Furthermore, no increase in MEK phosphorylation was detected upon ONOO(-) treatment. Staurosporine was used to investigate whether protein kinase C (PKC) is involved. This was confirmed by down-regulation of PKC by phorbol-12,13-dibutyrate, which resulted in significant reduction of ERK1/2 phosphorylation by ONOO(-), implying that activation of ERK by ONOO(-) depends on activation of PKC. Indeed, PKCalpha and epsilon were activated upon ONOO(-) exposure. When cells were treated with ONOO(-) in a calcium-free buffer, no activation of PKCalpha was detected. Concomitantly, a reduction of ERK1/2 phosphorylation was observed suggesting that calcium was required for translocation of PKCalpha and ERK phosphorylation by ONOO(-). Indeed, ONOO(-) exposure resulted in increased cytosolic calcium, which depended on the presence of extracellular calcium. Finally, data using G?6976, an inhibitor of calcium-dependent PKC activation, implied that ONOO(-)-mediated ERK1/2 phosphorylation depends on activation of a calcium-dependent PKC.