Mitogen-activated Protein Kinase Activation In Cells Research Articles

Transduced PEP-1-FK506BP Ameliorates Atopic Dermatitis in NC/Nga Mice

Immunophilin, FK506-binding protein 12 (FK506BP), is a receptor protein for the immunosuppressive drug FK506 by the FK506BP/FK506 complex. However, the precise function of FK506BP in inflammatory diseases remains unclear. Therefore, we examined the protective effects of FK506BP on atopic dermatitis (AD) in tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ)-induced HaCaT cells and 2,4-dinitrofluorobenzene-induced AD-like dermatitis in Nishiki-nezumi Cinnamon/Nagoya (NC/Nga) mice using a cell-permeable PEP-1-FK506BP. Transduced PEP-1-FK506BP significantly inhibited the expression of cytokines, as well as the activation of NF-κB and mitogen-activated protein kinase (MAPK) in TNF-α/IFN-γ-induced HaCaT cells. Furthermore, topical application of PEP-1-FK506BP to NC/Nga mice markedly inhibited AD-like dermatitis as determined by a histological examination and assessment of serum IgE levels, as well as cytokines and chemokines. These results indicate that PEP-1-FK506BP inhibits NF-κB and MAPK activation in cells and AD-like skin lesions by reducing the expression levels of cytokines and chemokines, thus suggesting that PEP-1-FK506BP may be a potential therapeutic agent for AD.

Analysis of mitogen-activated protein kinase activation and interactions with regulators and substrates

Mitogen-activated protein kinase (MAPK) cascades are ubiquitous signal transduction modules in eukaryotes that are of great interest and importance. Here, we summarize some useful methods for the analysis of MAPK signaling, including methods to (1) detect MAPK activation in cells, with an emphasis on using phosphorylation-state-specific antibodies raised against mammalian phosphopeptide sequences to detect the activation of MAPKs in other species; (2) estimate the cellular concentrations of MAPKs and other proteins of interest; (3) detect and quantify the stable physical association of MAPKs with their substrates and regulators, and estimate the relevant dissociation constants; (4) delineate the MAPK-binding regions or domains of MAPK-interacting proteins, with particular emphasis on the identification and verification of MAPK-docking sites. These procedures are broadly applicable to many organisms, including both yeast and mammalian cells.

MAP kinase activation in cells exposed to a 60 Hz electromagnetic field.

This research provides evidence that mitogen-activated protein kinase or extracellular signal-regulated kinase (MAPK/ERK) is activated in HL-60 human leukemia cells, MCF-7 human breast cancer cells, and rat fibroblast 3Y1 cells exposed to a 60 Hertz (Hz), 1 Gauss (G) electromagnetic field (EMF). The effects of EMF exposure were compared to those observed using 12-O-tetradecanoylphorbal-13-acetate (TPA) treatment. The level of MAPK activation in cells exposed to EMF was approximately equivalent to that in cells treated with 0.1-0.5 ng/ml of TPA. A role for protein kinase C (PKC) in the process leading to MAPK activation in EMF exposed cells is also suggested by the results. MAPK activation is negated by an inhibitor to PKCalpha, but not PKCdelta inhibitors, in cells subjected to EMF exposure or TPA treatment. Thus, similarities between the effects of EMF exposure and TPA treatment are supported by this investigation. This provides a possible method for revealing other participants in EMF-cell interaction, since the TPA induction pathway is well documented.

Mechanisms of H4/ICOS costimulation: effects on proximal TCR signals and MAP kinase pathways.

H4/ICOS is a costimulatory molecule related to CD28. Its effects on early TCR signals have been analyzed in mouse CD4(+) Th2 cells, expressing H4/ICOS at higher levels than Th1 clones. Anti-H4/ICOS antibodies strongly enhanced CD3-mediated tyrosine phosphorylation of ZAP-70, zeta, or Vav, as well as extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 MAP kinase activation in these cells. The association of phosphoinositide 3-kinase (PI-3K) to H4/ICOS was enhanced by H4/ICOS cross-linking, and PI-3K inhibitors inhibited ERK and JNK activation and IL-4/IL-10 secretion, but not p38 MAP kinase or ZAP-70 activation. H4/ICOS-mediated activation of JNK, but not ERK or p38, is partially dependent on the expression of CD4 by the cells, whereas H4/ICOS costimulation is partially independent on CD28 expression. Cytochalasin D, an inhibitor of actin polymerization, inhibited ZAP-70, MAP kinase activation, or IL-4/IL-10 secretion. Neither cyclosporin A nor inhibitors of PKC produced detectable inhibition of ZAP-70 phosphorylation or MAP kinase activation in these Th2 cells. Cyclosporin A strongly inhibited IL-4, but not IL-10 secretion. ERK or JNKinhibitors partially inhibited IL-4 and IL-10 secretion, while PKC or p38 inhibitors had no significant effects on IL-4 or IL-10 secretion. Taken together, our data show clear similarities of costimulation mechanisms between H4/ICOS and CD28 during the early steps of TCR activation.

Chronic exposure of human mesangial cells to high glucose environments activates the p38 MAPK pathway

High glucose (HG) environments activate several protein kinase pathways in mesangial cells, including the mitogen-activated protein kinase (MAPK) pathway, ERK. The p38 MAPK pathway is activated by events that occur in the setting of diabetes, such as protein kinase C (PKC) up-regulation and cellular stresses (osmotic stress and redox changes). Substrates of activated p38 MAPK include transcription factors that are involved in the microvascular complications of diabetes. This current study investigated the mechanisms of HG-mediated activation of p38 MAPK in cultured human mesangial cells (HMCs) and the effects of p38 MAPK activation on the transcription factor activator protein-1 (AP-1). HMCs were cultured in 5 mmol/L D-glucose [normal glucose (NG)] or 30 mmol/L D-glucose (HG) for seven days. Cells were also treated with HG for brief periods of time (0.5 to 4 hours) to assess the acute effects of HG on p38 MAPK. Using Western blotting of HMC lysates, changes in the tyrosine and threonine phosphorylation of p38 MAPK were measured. The kinase activity of immunoprecipitated p38 MAPK was determined by an in vitro assay that measured the phosphorylation and activation of MAPKAP kinase-2, an intermediary signaling protein downstream of p38 MAPK. To investigate the role of osmotic stress in HG activation of p38 MAPK, cells were acutely treated with mannitol (25 to 250 mOsm/L x 5 to 60 min) or were grown seven days in media supplemented with mannitol at concentrations iso-osmotic to HG media. To investigate the role of PKC in HG-mediated p38 MAPK activation, HMCs were treated with the PKC inhibitors GF 109203X, Ro 32-0432, or rottlerin during the last several hours of HG treatment. HG conditioned cells were also treated with the antioxidants L-N-acetylcysteine (L-NAC) or diphenyliodonium (DPI) prior to harvest. To determine a functional significance of HG-mediated p38 MAPK activation, the DNA binding of the transcription factor complex AP-1 was measured by electrophoretic mobility shift assay. The p38 MAPK pathway was not activated by the acute addition of HG to the HMCs. However, activation of p38 MAPK in HMCs grown seven days in HG was demonstrated by increased tyrosine and threonine phosphorylation of p38 MAPK proteins and increased kinase activity of immunoprecipitated p38 MAPK. As assessed by a kinase assay, p38 MAPK activity in cells grown in HG for seven days exceeded that of NG cells by more than 250%. This difference was not due to differences in the amount of p38 MAPK protein between the treatment groups. Acute osmotic activation of p38 MAPK occurred at extremely high mannitol concentrations (250 mOsm/L) that exceeded the osmotic stress of acute HG. Furthermore, in cells grown for seven days in mannitol at concentrations similar to HG, p38 MAPK activity was similar to control values. Phorbol ester (PMA) treatment stimulated a twofold increase in p38 MAPK activity. The addition of GFX or Ro 32-0432 to HG cells, at concentrations that inhibited PMA activation of p38 MAPK, did not inhibit the glucose-mediated p38 MAPK activation. Rottlerin, a PKC delta inhibitor, also failed to reverse the HG-mediated p38 MAPK activation. Treatment of HG cells with L-NAC or DPI inhibited the HG-mediated p38 MAPK phosphorylation. As we have previously shown, DNA binding of the transcription factor complex AP-1 was increased in HG cells. This binding was reversed by treatment of the HG cells with the p38 MAPK inhibitor SB 203580. Chronic exposure of HMC to HG concentrations activates the p38 MAPK pathway. This activation appears to be independent of changes in the amount of total p38 MAPK produced by the cells, independent of chronic osmotic stress and independent of PKC activation. The reversal of p38 MAPK by L-NAC and DPI suggests the glucose-mediated p38 MAPK activation may occur via reactive oxygen species. The activity of AP-1, a transcription factor complex that regulates several genes involved in diabetic nephropathy, is reversed when the p38 MAPK pathway is inhibited. These findings suggest the p38 MAPK pathway may be an important pathway involved in diabetic complications.

Insulin receptor substrate-1-mediated enhancement of growth hormone-induced mitogen-activated protein kinase activation.

Interaction of GH with the cell-surface GH receptor (GHR) causes activation of the GHR-associated tyrosine kinase, JAK2, and consequent triggering of signaling cascades including the STAT, Ras/Raf/MEK1/MAP kinase, and insulin receptor substrate-1(IRS-1)/PI3kinase pathways. We previously showed that IRS- and GHR-deficient 32D cells that stably express the rabbit GHR and rat IRS-1 (32D-rbGHR-IRS-1) exhibited markedly enhanced GH-induced proliferation and MAP kinase (ERK1 and ERK2) activation compared with cells expressing only the GHR (32D-rbGHR). We now examine biochemical mechanism(s) by which IRS-1 augments GH-induced MAP kinase activation. Time-course experiments revealed a similarly transient (maximal at 15 min) GH-induced ERK1 and ERK2 activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells, but, consistent with our prior findings, substantially greater activation was seen in the IRS-1-containing cells. In both cells, GH-induced MAP kinase activation was markedly blunted by the MEK1 inhibitor, PD98059, but not by the PKC inhibitor, GF109203X. Interestingly, pretreatment with the PI3K inhibitor, wortmannin (EC50 approximately 10 nM), significantly reduced GH-induced MAP kinase activation in both 32D-rbGHR and 32D-rbGHR-IRS-1 cells. This same pattern in both cells of IRS-1-dependent augmentation and IRS-1-independent wortmannin sensitivity was also observed for GH-induced activation of Akt and MEK1 (using state-specific antibody blotting for both), despite the lack of difference in GHR, JAK2, SHP-2, p85, Akt, Ras, Raf-1, MEK1, ERK1, or ERK2 abundance between the two cells. A different PI3K inhibitor, LY294002 (50 microM), substantially inhibited (roughly 72%) GH-induced MAP kinase activation in 32D-rbGHR-IRS-1 cells, but only marginally (and statistically insignificantly) inhibited GH-induced MAP kinase activation in 32D-rbGHR cells. Because GH-induced Akt activation was completely inhibited in both cells by the same concentration of LY294002, these findings indicate that the wortmannin sensitivity of both the IRS-1-independent and -dependent GH-induced MAP kinase activation may reflect the activity of another wortmannin-sensitive target(s) in addition to PI3K in mediation of GH-induced MAP kinase activation in these cells. Notably, GH-induced STAT5 tyrosine phosphorylation, unlike Akt or MAPK activation, did not differ between the cells. Finally, while GH promoted accumulation of activated Ras in both cells, both basal and GH-induced activated Ras levels were greater in cells expressing IRS-1 than in 32D-rbGHR cells. These data indicate that while GH induces tyrosine phosphorylation of STAT5 and activation of the Ras/Raf/MEK1/MAPK and PI3K pathways, IRS-1 expression augments the latter two more than the former.

The Tyrosines in the Bidentate Motif of the env-sea Oncoprotein Are Essential for Cell Transformation and Are Binding Sites for Grb2 and the Tyrosine Phosphatase SHP-2

The transforming gene product of the S13 avian erythroblastosis virus, the env-sea protein, is a member of the hepatocyte growth factor receptor family of tyrosine kinases comprising Met, Ron, and Sea. Like all three members of this family, the env-sea protein has a so-called bidentate motif (Y557INMAVTY564VNL) composed of two tandemly arranged tyrosines in the carboxyl terminus. To investigate whether the tyrosine residues in this motif are essential for the env-sea-mediated transformation, we generated tyrosine to phenylalanine mutations. Substitutions of both tyrosine residues resulted in complete loss of the transforming activity. In contrast, single mutations at either tyrosine did not inhibit transformation of Rat1 cells, and mutation of tyrosine 564 actually increased transformation of Rat 1 cells. To define signaling pathways activated by the env-sea protein, we looked for protein-protein interactions mediated by these tyrosine residues. We show that the bidentate motif is responsible for interaction with the adapter protein Grb2, phosphatidylinositol 3-kinase, and the tyrosine phosphatase SHP-2. Furthermore, we show that microinjected Src homology 2 domains from either Grb2 or SHP-2 blocked the transforming activity of the env-sea protein. Together, these results suggest that the tyrosines within the bidentate motif are essential for the env-sea transformation.

Enhancement of EGF- and PMA-mediated MAP kinase activation in cells expressing the human papillomavirus type 16 E5 protein.

In this report we demonstrate that cells expressing the human papillomavirus type 16 E5 open reading frame (HPV16-E5) show a greatly enhanced transcription of the immediate early genes after EGF or PMA treatment compared to control cells. This enhancement is due to amplification of the signal transduction pathways in response to growth factors or phorbol esters. Upon short-time EGF treatment of the E5-expressing cells we observed an increase in the activation of EGF receptors, resulting in a stronger activation of MAP kinases ERK1/2 compared to control-transfected cells. We also observed that in E5-expressing cells, treatment with PMA results in an increase in membrane-associated PKC activity, and a superactivation of the ERK1/2 MAP kinases. This superactivation is PKC-dependent, since pretreatment of the cells with the PKC inhibitor Ro 31-8220 inhibits MAP kinase activation and early gene transcription almost completely. Furthermore, treatment with genistein strongly reduces the PMA-mediated superactivation of ERK1/2 kinases, demonstrating a PKC-mediated, tyrosine kinase-dependent pathway in the superinduction of MAP kinase activation. Thus, HPV16-E5 effects superactivation of MAP kinases over at least two different pathways, a PKC-mediated, and another, receptor tyrosine-kinase mediated, PKC-independent one.

Dependence of Activated Gα12-induced G1 to S Phase Cell Cycle Progression on Both Ras/Mitogen-activated Protein Kinase and Ras/Rac1/Jun N-terminal Kinase Cascades in NIH3T3 Fibroblasts

We evaluated the roles of mitogen-activated protein kinase (MAPK) and Jun N-terminal kinase (JNK) signaling cascades in Galpha12-induced G1 to S phase cell cycle progression in NIH3T3(M17) fibroblasts. Transient expression of a constitutively active mutant of Galpha12, Galpha12(R203C), resulted in a 2-fold increase in the number of bromodeoxyuridine-positive S phase cells over vector control level under serum-deprived conditions. Consistent with the ability of Galpha12(R203C) to induce G1/S transition, its expression led to a 2-fold increase in cyclin A promoter activity, which showed a marked synergism with a low concentration of serum, resulting in up to a 15-fold elevation over the basal level. In addition, Galpha12(R203C) caused a 2-fold stimulation in E2F-mediated transactivation. Wild type Galpha12 showed similar stimulatory effects on cyclin A promoter activity and E2F-mediated transactivation, although of lesser magnitude. We observed a modest but constitutive activation of MAPK in cells transfected with Galpha12(R203C), which was abolished by a dominant negative form of Ras. Galpha12(R203C) also induced a 3-fold increase in JNK activity, which was abolished by dominant negative forms of either Rac1 or Ras. The expression of dominant negative forms of Ras, MAPK, Rac1, or JNK inhibited Galpha12(R203C)-induced increases in bromodeoxyuridine-positive cells. Also, the dominant negative forms of Ras, MAPK, and JNK strongly inhibited Galpha12(R203C)-induced stimulation of cyclin A promoter activity. These results demonstrate that both the Ras/MAPK and Ras/Rac1/JNK pathways convey necessary, if not sufficient, mitogenic signals induced by Galpha12 activation.

1-O-octadecyl-2-O-methyl-glycerophosphocholine inhibits the transduction of growth signals via the MAPK cascade in cultured MCF-7 cells.

1-O-Octadecyl-2-O-methyl-glycerophosphocholine (ET18-OCH3) is an ether lipid with selective antiproliferative properties whose mechanism of action is still unresolved. We hypothesized that since ET18-OCH3 affects a wide variety of cells, its mechanism of action was likely to involve the inhibition of a common widely used pathway for transducing growth signals such as the mitogen-activated protein kinase (MAPK) cascade. To test this, we established conditions whereby quiescent MCF-7 cells took up ET18-OCH3 in sufficient quantities that inhibited cell proliferation subsequent to the addition of growth medium and examined the activation of components of the MAPK cascade under these conditions. ET18-OCH3 inhibited the sustained phosphorylation of MAPK resulting in a decrease in the magnitude and duration of activation of MAPK in cells stimulated with serum or EGF. ET18-OCH3 had no effect on the binding of EGF to its receptors, their activation, or p21ras activation. However, an interference in the association of Raf-1 with membranes and a resultant decrease in Raf-1 kinase activity in membranes of ET18-OCH3-treated cells was observed. ET18-OCH3 had no direct effect on MAPK or Raf-1 kinase activity. A direct correlation between ET18-OCH3 accumulation, inhibition of cell proliferation, Raf association with the membrane, and MAPK activation was also established. These results suggest that inhibition of the MAPK cascade by ET18-OCH3 as a result of its effect on Raf-1 activation may be an important mechanism by which ET18-OCH3 inhibits cell proliferation.

Altered expression of protein-tyrosine phosphatase 2C in 293 cells affects protein tyrosine phosphorylation and mitogen-activated protein kinase activation.

PTP2C, an SH2 domain-containing protein-tyrosine phosphatase, is recruited to the growth factor receptors upon stimulation of cells. To investigate its role in growth factor signaling, we have overexpressed by approximately 6-fold the native PTP2C and a catalytically inactive mutant of the enzyme in 293 human embryonic kidney cells. The native PTP2C was located entirely in the cytosol, while the inactive mutant was nearly equally distributed in cytsolic and membrane fractions. Expression of the latter caused hyperphosphorylation on tyrosine of a 43-kDa protein, which was coimmunoprecipitated and co-partitioned in the plasma membrane fraction with the inactive PTP2C mutant. This protein may represent a physiological substrate of PTP2C. Overexpression of the native PTP2C enhanced epidermal growth factor (EGF)-stimulated mitogen-activated protein (MAP) kinase activity by 30%, whereas expression of the inactive mutant reduced the stimulated activity by 50%. Similar effects were observed for the activation of MAP kinase as determined by activity assay, gel mobility shift, and tyrosine phosphorylation. The data suggest that the phosphatase activity of PTP2C is partly required for MAP kinase activation by EGF and that PTP2C may function by dephosphorylating the 43-kDa membrane protein.