The death domain kinase RIP1 is essential for tumor necrosis factor alpha signaling to p38 mitogen-activated protein kinase

The cytokine tumor necrosis factor alpha (TNF-alpha) stimulates the NF-kappaB, SAPK/JNK, and p38 mitogen-activated protein (MAP) kinase pathways by recruiting RIP1 and TRAF2 proteins to the tumor necrosis factor receptor 1 (TNFR1). Genetic studies have revealed that RIP1 links the TNFR1 to the IkappaB kinase (IKK) complex, whereas TRAF2 couples the TNFR1 to the SAPK/JNK cascade. In transfection studies, RIP1 and TRAF2 stimulate p38 MAP kinase activation, and dominant-negative forms of RIP1 and TRAF2 inhibit TNF-alpha-induced p38 MAP kinase activation. We found TNF-alpha-induced p38 MAP kinase activation and interleukin-6 (IL-6) production impaired in rip1(-/-) murine embryonic fibroblasts (MEF) but unaffected in traf2(-/-) MEF. Yet, both rip1(-/-) and traf2(-/-) MEF exhibit a normal p38 MAP kinase response to inducers of osmotic shock or IL-1alpha. Thus, RIP1 is a specific mediator of the p38 MAP kinase response to TNF-alpha. These studies suggest that TNF-alpha-induced activation of p38 MAP kinase and SAPK/JNK pathways bifurcate at the level of RIP1 and TRAF2. Moreover, endogenous RIP1 associates with the MAP kinase kinase kinase (MAP3K) MEKK3 in TNF-alpha-treated cells, and decreased TNF-alpha-induced p38 MAP kinase activation is observed in Mekk3(-/-) cells. Taken together, these studies suggest a mechanism whereby RIP1 may mediate the p38 MAP kinase response to TNF-alpha, by recruiting the MAP3K MEKK3.     

Hypoxia-induced proliferative response of vascular adventitial fibroblasts is dependent on g protein-mediated activation of mitogen-activated protein kinases

Hypoxia has been shown to act as a proliferative stimulus for adventitial fibroblasts of the pulmonary artery. The signaling pathways involved in this growth response, however, remain unclear. We tested the hypothesis that hypoxia-induced proliferation of fibroblasts would be dependent on distinct (compared with serum) activation and utilization patterns of mitogen-activated protein (MAP) kinases initiated by Galpha(i/o) proteins. We found that hypoxia stimulated increases in DNA synthesis and growth of quiescent fibroblasts in the absence of exogenous mitogens and also markedly augmented serum-stimulated growth responses. Hypoxia caused a transient activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), the time course and pattern of which was somewhat similar to that induced by serum but which was of lesser magnitude. On the other hand, hypoxia-induced activation of p38 MAP kinase was biphasic, whereas serum-stimulated activation of p38 MAP kinase was transient, and the magnitude of activation was greater for hypoxia compared with that of serum stimulation. ERK1/2, JNK1, and p38 MAP kinase but not JNK2 were necessary for hypoxia-induced proliferation because PD98059, SB202190, and JNK1 antisense oligonucleotides nearly ablated the growth response. JNK2 appeared to act as a negative modulator of hypoxia-induced growth because JNK2 antisense oligonucleotides led to an increase in DNA synthesis. In serum-stimulated cells, antisense JNK1 oligonucleotides and PD98059 had inhibitory effects on proliferation, whereas SB202190 led to an increase in DNA synthesis. Pertussis toxin, which blocks Galpha(i/o)-mediated signaling, markedly attenuated hypoxia-induced DNA synthesis and activation of ERK and JNK but not p38 MAP kinase. We conclude that hypoxia itself can act as a growth promoting stimulus for subsets of bovine neonatal adventitial fibroblasts largely through Galpha(i/o)-mediated activation of a complex network of MAP kinases whose specific contributions to hypoxia-induced proliferation differ from traditional serum-induced growth signals.     

The p42/p44 mitogen-activated protein kinase activation triggers p27Kip1 degradation independently of CDK2/cyclin E in NIH 3T3 cells.

The p42/p44 mitogen-activated protein (MAP) kinase is stimulated by various mitogenic stimuli, and its sustained activation is necessary for cell cycle G(1) progression and G(1)/S transition. G(1) progression and G(1)/S transition also depend on sequential cyclin-dependent kinase (CDK) activation. Here, we demonstrate that MAP kinase inhibition leads to accumulation of the CDK inhibitor p27(Kip1) in NIH 3T3 cells. Blocking the proteasome-dependent degradation of p27(Kip1) impaired this accumulation, suggesting that MAP kinase does not act on p27(Kip1) protein synthesis. In the absence of extracellular signals (growth factors or cell adhesion), genetic activation of MAP kinase decreased the expression of p27(Kip1) as assessed by cotransfection experiments and by immunofluorescence detection. Importantly, MAP kinase activation also decreased the expression of a p27(Kip1) mutant, which cannot be phosphorylated by CDK2, suggesting that MAP kinase-dependent p27(Kip1) regulation is CDK2-independent. Accordingly, expression of dominant-negative CDK2 did not impair the down-regulation of p27(Kip1) induced by MAP kinase activation. These data demonstrate that the MAP kinase pathway regulates p27(Kip1) expression in fibroblasts essentially through a degradation mechanism, independently of p27(Kip1) phosphorylation by CDK2. This strengthens the role of this CDK inhibitor as a key effector of G(1) growth arrest, whose expression can be controlled by extracellular stimuli-dependent signaling pathways.     

p67/MetAP2 suppresses K-RasV12-mediated transformation of NIH3T3 mouse fibroblasts in culture and in athymic mice

In many tumor cells, the activation and activity of extracellular signal-regulated kinases (ERK1/2) are very high because of the constitutive activation of the Ras-mediated signaling pathway. Here, we ectopically expressed the human homologue of rat eukaryotic initiation factor 2-associated glycoprotein, p67/MetAP2, in EGF-treated mouse embryonic NIH3T3 fibroblasts and C2C12 myoblasts and NIH3T3 cell lines expressing the constitutively active form of MAP kinase kinase (MEK) to inhibit the activation and activity of ERK1/2 MAP kinases. In addition, we also ectopically expressed rat p67/MetAP2 in oncogenic Ras-induced transformed NIH3T3 fibroblasts and inhibited their transformed phenotype both in culture and in athymic nude mice possibly by inhibiting angiogenesis. This inhibition of ERK1/2 MAP kinases is due to the direct binding with rat p67/MetAP2, and this leads to the inhibition of activity of ERK1/2 MAP kinases both in vitro and in vivo. Furthermore, expression of p67/MetAP2 siRNA in both NIH3T3 fibroblasts and C2C12 myoblasts causes activation and activity of ERK1/2 MAP kinases. Our results thus suggest that ectopic expression of rat p67/MetAP2 in transformed cells can inhibit the tumorigenic phenotype by inhibiting the activation and activity of ERK1/2 MAP kinases and, thus, that p67/MetAP2 has tumor suppression activity.     

Gi-mediated activation of the Ras/MAP kinase pathway involves a 100 kDa tyrosine-phosphorylated Grb2 SH3 binding protein, but not Src nor Shc.

Mitogenic G protein-coupled receptors, such as those for lysophosphatidic acid (LPA) and thrombin, activate the Ras/MAP kinase pathway via pertussis toxin (PTX)-sensitive Gi, tyrosine kinase activity and recruitment of Grb2, which targets guanine nucleotide exchange activity to Ras. Little is known about the tyrosine phosphorylations involved, although Src activation and Shc phosphorylation are thought to be critical. We find that agonist-induced Src activation in Rat-1 cells is not mediated by Gi and shows no correlation with Ras/MAP kinase activation. Furthermore, LPA-induced tyrosine phosphorylation of Shc is PTX-insensitive and Ca2+-dependent in COS cells, but undetectable in Rat-1 cells. Expression of dominant-negative Src or Shc does not affect MAP kinase activation by LPA. Thus, Gi-mediated Ras/MAP kinase activation in fibroblasts and COS cells involves neither Src nor Shc. Instead, we detect a 100 kDa tyrosine-phosphorylated protein (p100) that binds to the C-terminal SH3 domain of Grb2 in a strictly Gi- and agonist-dependent manner. Tyrosine kinase inhibitors and wortmannin, a phosphatidylinositol (PI) 3-kinase inhibitor, prevent p100-Grb2 complex formation and MAP kinase activation by LPA. Our results suggest that the p100-Grb2 complex, together with an upstream non-Src tyrosine kinase and PI 3-kinase, couples Gi to Ras/MAP kinase activation, while Src and Shc act in a different pathway.     

Oncogenic ras and p53 cooperate to induce cellular senescence

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.     

Arachidonic acid activates mitogen-activated protein (MAP) kinase-activated protein kinase 2 and mediates adhesion of a human breast carcinoma cell line to collagen type IV through a p38 MAP kinase-dependent pathway

Adhesion of metastatic human mammary carcinoma MDA-MB-435 cells to the basement membrane protein collagen type IV can be activated by treatment with arachidonic acid. We initially observed that this arachidonic acid-mediated adhesion was inhibited by the tyrosine kinase inhibitor genistein. Therefore, we examined the role of the mitogen-activated protein (MAP) kinase family tyrosine phosphorylation-regulated pathways in arachidonic acid-stimulated cell adhesion. Arachidonic acid stimulated the phosphorylation of p38, the activation of MAP kinase-activated protein kinase 2 (MAPKAPK2, a downstream substrate of p38), and the phosphorylation of heat shock protein 27 (a downstream substrate of MAP kinase-activated protein kinase 2). Treatment with the p38 inhibitor PD169316 completely and specifically inhibited arachidonic acid-mediated cell adhesion to collagen type IV. p38 activity was specifically associated with arachidonic acid-stimulated adhesion; this was demonstrated by the observation that 12-O-tetradecanoylphorbol 13-acetate-activated cell adhesion was not blocked by inhibiting p38 activity. Extracellular signal-regulated protein kinases (ERKs) 1 and 2 were also activated by arachidonic acid; however, cell adhesion to collagen type IV was not highly sensitive to PD98059, an inhibitor of MAP kinase kinase/ERK kinase 1 (MEK1) that blocks activation of the ERKs. c-Jun NH(2)-terminal kinase was not activated by arachidonic acid treatment of these cells. Together, these data suggest a novel role for p38 MAP kinase in regulating adhesion of breast cancer cells to collagen type IV.     

Formation of reactive oxygen species in rat epithelial cells upon stimulation with fly ash

Fly ash was used as a model for ambient particulate matter which is under suspicion to cause adverse pulmonary health effects. The fly ash was pre-sized and contained only particles < 20 microm including an ultrafine fraction (< 100 nm) that contributed 31% to the particle number. In our study, we investigated the influence of fly ash on the promotion of early inflammatory reactions like the formation of reactive oxygen species (ROS) in rat lung epithelial cells (RLE-6TN). Furthermore, we determined the formation of nitric oxide (NO). The cells show a clear dose-response relationship concerning the formation of ROS with regard to the mass of particles applied. Lipopolysaccharide (LPS) added as a co-stimulus did not increase the formation of ROS induced by fly ash. Furthermore, in LPS (0.1 microg/ml) and tumour necrosis factor-alpha (TNF-alpha; 1 ng/ml) pre-treated cells no increase in reactive oxygen species comparable to fly ash alone is observable. In presence of the metal chelator, desferrioxamine (DFO), ROS formation can be significantly reduced. Neither fly ash nor LPS induced a significant NO release in RLE-6TN cells.     

Incinerator fly ash provokes alteration of redox equilibrium and liberation of arachidonic acid in vitro

Numerous epidemiological studies have associated exposure to ambient particulate matter (PM) with pulmonary and cardiovascular health effects. Macrophages as a part of the primary pulmonary defence system play a crucial role by generating pro- and anti-inflammatory mediators. The aim of the present study was to examine the effect of incinerator fly ash (MAF02) as a model of environmental particulate matter on the formation of reactive oxygen species (ROS) and their ability to induce oxidative stress in RAW264.7 macrophages. Furthermore, the liberation of arachidonic acid (AA) was observed. The interaction of MAF02 with macrophages caused increased mobilisation of AA, accompanied by enhanced expression of cyclooxygenase-2 (COX-2). The MAF02-induced AA liberation was found to depend on an increased intracellular calcium concentration. In addition, MAF02-induced liberation of AA was selectively blocked by an ERK1/2 pathway-specific inhibitor, while inhibition of the p38 MAPK activity had no effect. Fly ash was also observed to induce an increase in cellular glutathione (GSH) content and antioxidative enzyme haem oxygenase-1 (HO-1). In correlation, experiments with dichlorofluorescein demonstrated increased formation of ROS upon treatment with fly ash. In summary, incinerator fly ash induces oxidative stress to a certain extent, resulting in the onset of important mechanisms related to inflammation.     

Ethanol potentiates the mitogenic effects of sphingosine 1-phosphate by a zinc- and calcium-dependent mechanism in fibroblasts.

In mouse embryo NIH 3T3 fibroblasts, ethanol (60-80 mM) was found to enhance the stimulatory effects of sphingosine 1-phosphate (S1P) on both DNA synthesis and cell proliferation. Well-detectable potentiating effects of ethanol on S1P-induced mitogenesis required the presence of calcium (>1 mM) and zinc (20-40 microM) in the incubation medium. The amphibian tetrapeptide bombesin, which is known to mobilize intracellular calcium in fibroblasts, had no effect alone, but it approximately doubled the combined stimulatory effects of ethanol and S1P on DNA synthesis. The synergistic mitogenic effects of ethanol and S1P were also slightly enhanced, rather than inhibited, by the alcohol dehydrogenase inhibitor 4-methylpyrazole (5 mM). Of the various growth regulatory enzymes examined, ethanol detectably enhanced the stimulatory effects of S1P on the phosphosphorylation (activation) of p42/p44 mitogen-activated protein (MAP) kinases, but not of p38 MAP kinase. Cotreatment of fibroblasts with ethanol for 10 min also enhanced the stimulatory effects of S1P on the activities of c-Raf-1 kinase and p70 S6 kinase, but neither S1P nor ethanol had effects on phosphatidylinositol 3'-kinase and Akt/PKB kinase activities. Ethanol-plus-S1P-induced DNA synthesis was partially inhibited by both PD 98059 (50 microM) and rapamycin (10 nM), inhibitors of p42/p44 MAP kinase kinase and mTOR/p70 S6 kinases, respectively. The results indicate that in NIH 3T3 fibroblasts, ethanol can enhance the mitogenic effects of S1P by a zinc- and calcium-dependent mechanism involving both the rapamycin-sensitive p70 S6 kinase-dependent and the c-Raf-1/MAP kinase-dependent growth regulatory pathways.     

p42/p44 MAP kinase activation is localized to caveolae-free membrane domains in airway smooth muscle

Caveolae are abundant plasma membrane invaginations in airway smooth muscle that may function as preorganized signalosomes by sequestering and regulating proteins that control cell proliferation, including receptor tyrosine kinases (RTKs) and their signaling effectors. We previously demonstrated, however, that p42/p44 MAP kinase, a critical effector for cell proliferation, does not colocalize with RTKs in caveolae of quiescent airway myocytes. Therefore, we investigated the subcellular sites of growth factor-induced MAP kinase activation. In quiescent myocytes, though epidermal growth factor receptor (EGFR) was almost exclusively found in caveolae, p42/p44 MAP kinase, Grb2, and Raf-1 were absent from these membrane domains. EGF induced concomitant phosphorylation of caveolin-1 and p42/p44 MAP kinase; however, EGF did not promote the localization of p42/p44 MAP kinase, Grb2, or Raf-1 to caveolae. Interestingly, stimulation of muscarinic M(2) and M(3) receptors that were enriched in caveolae-deficient membranes also induced p42/p44 MAP kinase phosphorylation, but this occurred in the absence of caveolin-1 phosphorylation. This suggests that the localization of receptors to caveolae and interaction with caveolin-1 is not directly required for p42/p44 MAP kinase phosphorylation. Furthermore, we found that EGF exposure induced rapid translocation of EGFR from caveolae to caveolae-free membranes. EGFR trafficking coincided temporally with EGFR and p42/p44 MAP kinase phosphorylation. Collectively, this indicates that although caveolae sequester some receptors associated with p42/p44 MAP kinase activation, the site of its activation is associated with caveolae-free membrane domains. This reveals that directed trafficking of plasma membrane EGFR is an essential element of signal transduction leading to p42/p44 MAP kinase activation.     

Cell type-specific effect of hypoxia and platelet-derived growth factor-BB on extracellular matrix turnover and its consequences for lung remodeling

Hypoxia is associated with extracellular matrix remodeling in several inflammatory lung diseases, such as fibrosis, chronic obstructive pulmonary disease, and asthma. In a human cell culture model, we assessed whether extracellular matrix modification by hypoxia and platelet-derived growth factor (PDGF) involves the action of matrix metalloproteinases (MMPs) and thereby affects cell proliferation. Expression of MMP and its activity were assessed by zymography and enzyme-linked immunosorbent assay in human lung fibroblasts and pulmonary vascular smooth muscle cells (VSMCs), and synthesis of soluble collagen type I was assessed by enzyme-linked immunosorbent assay. In both cell types, hypoxia up-regulated the expression of MMP-1, -2, and -9 precursors without subsequent activation. MMP-13 was increased by hypoxia only in fibroblasts. PDGF-BB inhibited the synthesis and secretion of all hypoxia-dependent MMP via Erk1/2 mitogen-activated protein (MAP) kinase activation. Hypoxia and PDGF-BB induced synthesis of soluble collagen type I via Erk1/2 and p38 MAP kinase. Hypoxia-induced cell proliferation was blocked by antibodies to PDGF-BB or by inhibition of Erk1/2 but not by the inhibition of MMP or p38 MAP kinase in fibroblasts. In VSMCs, hypoxia-induced proliferation involved Erk1/2 and p38 MAP kinases and was further increased by fibroblast-conditioned medium or soluble collagen type I via Erk1/2. In conclusion, hypoxia controls tissue remodeling and proliferation in a cell type-specific manner. Furthermore, fibroblasts may affect proliferation of VSMC indirectly by inducing the synthesis of soluble collagen type I.     

Structural and Functional Basis for p38-MK2-Activated Rsk Signaling in Toll-Like Receptor-Stimulated Dendritic Cells

Rsk kinases play important roles in several cellular processes such as proliferation, metabolism, and migration. Until recently, Rsk activation was thought to be exclusively initiated by Erk1/2, but in dendritic cells (DC) Rsk is also activated by p38 mitogen-activated protein (MAP) kinase via its downstream substrates, MK2/3. How and why this noncanonical configuration of the MAP kinase pathway is adopted by these key immune cells are not known. We demonstrate that the Erk1/2-activated C-terminal kinase domain of Rsk is dispensable for p38-MK2/3 activation and show that compared with fibroblasts, a greater fraction of p38 and MK2/3 is located in the cytosol of DC prior to stimulation, suggesting a partial explanation for the operation of the noncanonical pathway of Rsk activation in these cells. p38/MK2/3-activated Rsk phosphorylated downstream targets and is physiologically important because in plasmacytoid DC (pDC) stimulated with Toll-like receptor 7 (TLR7) agonists, Erk1/2 activation is very weak relative to p38. As a result, Rsk activation is entirely p38 dependent. We show that this unusual configuration of MAP kinase signaling contributes substantially to production of type I interferons, a hallmark of pDC activation.     

The differential interaction of p38 MAP kinase and tumor necrosis factor-alpha in human alveolar macrophages and monocytes induced by Mycobacterium tuberculois

Cellular signaling by TNF-alpha is mediated through activation of mitogen activated protein (MAP) kinases. In particular, p38 MAP kinase is activated in mononuclear phagocytes and may be important in sustaining TNF-alpha activity. Here, we compared the activation and mutual regulation of p38 MAP kinase and TNF-alpha by MTB in human alveolar macrophages (AM) and blood monocytes (MN). AM and autologous MN were prepared, and stimulated by MTB at 1:1 (bacteria/cell). MAP kinase activation was assessed by immunoprecipitation and kinase activity. TNF-alpha mRNA was assessed by real-time RT-PCR, and TNF-alpha immunoreactivity was assessed by ELISA. MTB-induced p38MAP kinase rapidly in AM as compared to MN, and inhibition of p38 MAP kinase by SB203580 reduced both TNF-alpha mRNA and protein. Activation of ERK (1/2) by MTB followed similar kinetics in both AM and MN. TNF-alpha produced by MTB sustained p38 MAP kinase activation in MN only. These data suggest that interaction of resident pulmonary macrophages and the more immature MN with MTB differ with regard to both p38 MAP kinase activation and TNF-alpha expression.     

Hypochlorous acid stimulation of the mitogen-activated protein kinase pathway enhances cell survival

We investigated the activation of three subfamilies of mitogen-activated protein kinases (MAP kinase), the extracellular regulated kinase (ERK1/2), p38, and c-Jun N-terminal kinase (JNK), by the myeloperoxidase-derived oxidant HOCl, in human umbilical vein endothelial cells (HUVEC) and human skin fibroblasts. Treatment of fibroblasts with 10-30 microM HOCl induced a dose-dependent increase in the tyrosine phosphorylation of several proteins. ERK1/2 was activated by exposure to sublethal concentrations of reagent HOCl or by HOCl generated by myeloperoxidase as shown by immune complex kinase assays. Maximum activation was seen at 20 microM and peak activation occurred within 10 min. Western blot analysis demonstrated activation of p38 with 30 microM HOCl, occurring at 15-30 min. No activation of JNK was detected in the concentration range investigated. These results show that HOCl is able to activate MAP kinases. Effective doses were considerably lower than with H2O2 and the lack of JNK activation contrasts with the activation frequently seen with H2O2. Exposure to HOCl caused a loss of viability in HUVEC that was markedly enhanced when ERK1/2 activation was inhibited by U0126. This suggests that the activation of ERK promotes cell survival in response to the oxidative challenge.     

ATR-dependent activation of p38 MAP kinase is responsible for apoptotic cell death in cells depleted of Cdc7

Cdc7 is a serine/threonine kinase that plays essential roles in the initiation of eukaryotic DNA replication and checkpoint response. In previous studies, depletion of Cdc7 by small interfering RNA was shown to induce an abortive S phase that led to the cell cycle arrest in normal human fibroblasts and apoptotic cell death in various cancer cells. Here we report that stress-activated p38 MAP kinase was activated and responsible for apoptotic cell death in Cdc7-depleted HeLa cells. The activation of p38 MAP kinase in the Cdc7-depleted cells was shown to depend on ATR, a major sensor kinase for checkpoint or DNA damage responses. Only the p38 MAP kinase, and not the other stress-activated kinases such as JNK or ERK, was activated, and both caspase 8 and caspase 9 were activated for the induction of apoptosis. Activation of apoptosis in Cdc7-depleted cells was completely abolished in cells treated with small interfering RNA or an inhibitor of the p38 MAP kinase, suggesting that p38 MAP kinase activation was responsible for apoptotic cell death. Taken together, we suggest that the ATR-dependent activation of the p38 MAP kinase is a major signaling pathway that induces apoptotic cell death after depletion of Cdc7 in cancer cells.     

Activation of mitogen activated protein (MAP) kinases by vanadate is independent of insulin receptor autophosphorylation

Treatment of Chinese hamster ovary (CHO) cells over-expressing the human insulin receptor (CHO-HIRc) with the insulin mimetic agent, vanadate, resulted in a dose- and time-dependent tyrosine phosphorylation of two proteins with apparent molecular sizes of 42 kDa (p42) and 44 kDa (p44). However, vanadate was unable to stimulate the tyrosyi phosphorylation of theβ-subunit of the insulin receptor. By using myelin basic protein (MBP) as the substrate to measure mitogen-activated protein (MAP) kinase activity in whole cell lysates, vanadate-stimulated tyrosyl phosphorylation of p42 and p44 was associated with a dose- and time-dependent activation of MAP kinase activity. Furthermore, affinity purification of cell lysates on anti-phosphotyrosine agarose column followed by immunoblotting with a specific antibody to MAP kinases demonstrated that vanadate treatment increased the tyrosyl phosphorylation of both p44^mapk and p42^mapk by several folds, as compared to controls, in concert with MAP kinase activation. In addition, retardation in gel mobility further confirmed that vanadate treatment increased the phosphorylation of p44^mapk and p42^mapk in CHO-HIRc. A similar effect of vanadate on MAP kinase tyrosyl phosphorylation and activation was also observed in CHO cells over-expressing a protein tyrosine kinase-deficient insulin receptor (CHO-1018). These results demonstrate that the protein tyrosine kinase activity of the insulin receptor may not be required in the signaling pathways leading to the vanadate-mediated tyrosyl phosphorylation and activation of MAP kinases.     

Integration of multiple downstream signals determines the net effect of insulin on MAP kinase vs. PI 3'-kinase activation: potential role of insulin-stimulated H(2)O(2)

Cellular insulin stimulation generates a burst of H(2)O(2) that modulates protein-tyrosine phosphorylation in the insulin action pathway, in part by the inhibition of redox-sensitive protein-tyrosine phosphatases [J. Biol. Chem. 276 (2001) 21938]. Blocking the insulin-induced rise in H(2)O(2) with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) strongly attenuated the activation of phosphatidylinositol 3' (PI 3')-kinase, Akt and GLUT4 translocation by insulin in 3T3-L1 adipocytes; however, under identical conditions, we observed a paradoxical increase in the activation of p42/p44 mitogen-activated protein (MAP) kinase. DPI inhibited the insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1/2, and also reduced the association of Grb2 with IRS-1, suggesting that the effect of DPI on MAP kinase activation occurred downstream of the IR and IRS proteins. DPI increased the insulin-stimulated phosphorylation of p42/p44 MAP kinase with no change in basal, and increased insulin-stimulated MAP kinase kinase (MEK) activity by a similar degree. DPI enhanced basal Grb2-Sos binding and reduced the effect of insulin to potentiate the dissociation of the Grb2-Sos complex, suggesting that the effect of DPI was mediated upstream of Raf-1. Cell treatment with dibutyryl cAMP significantly reduced the enhancement of MAP kinase activation in the presence of DPI. However, forskolin, acting in a PKA-independent manner, increased the insulin stimulation of MAP kinase and MEK, but fully abrogated the effect of DPI to enhance these insulin responses. PLCgamma inhibition with U73122 blocked the insulin stimulation of MAP kinase and MEK as well as the enhancing effect of DPI on these responses. PKC activation strongly stimulated MAP kinase and MEK activation, even in the presence of U73122, consistent with PKC acting downstream of PLCgamma. These data show that the insulin-stimulated oxidant signal differentially affects the two major downstream components of the insulin signaling pathway, PI 3'-kinase and MAP kinase, and cross-talk between insulin action, PLCgamma and, to a lesser extent, PKA modulates the net cellular effects of insulin-stimulated cellular H(2)O(2).     

Farnesyl protein transferase inhibition interferes with activation of MAP kinase family members in human peripheral blood monocytes

BACKGROUND: Farnesyl protein transferase inhibitors have emerged as promising novel agents for combating cancerous disease. Nevertheless, the importance for farnesyl protein transferase enzymatic activity for cellular physiology of untransformed cells remains poorly investigated. MATERIALS AND METHODS: Peripheral blood monocytes, isolated from the blood of eight healthy volunteers, were treated with a farnesyl protein transferase inhibitor (FTI 744,832) or vehicle control for 16 hr. Subsequently cells were challenged with different concentrations of lipopolysaccharide (LPS), colony stimulating factor-1 (CSF-1), or phorbol esters for 10 min, after which the activation state of p42/p44 MAP kinase, p38 MAP kinase, and Jun-N-terminal kinase was investigated using Western blotting and phosphospecific antibodies. RESULTS: We observed that farnesyl protein transferase inhibition abrogated activation of p38 MAP kinase by LPS, CSF-1, and phorbol esters. Also the activation of Jun-N-terminal kinase by LPS was not seen after farnesyl protein transferase inhibition. Finally, stimulation of p42/p44 MAP kinase with CSF-1 was strongly reduced by farnesyl protein transferase inhibition, whereas activation of p42/p44 MAP kinase by phorbol ester was only slightly effected. CONCLUSIONS: Farnesyl protein transferase enzymatic activity is required for proper activation of all major members of the MAP kinase family. The observation that activation the p38 MAP kinase and Jun-N-terminal kinase is sensitive to farnesyl protein transferase inhibition raises the possibility that, in addition to cancerous disease, farnesyl protein transferase inhibitors may be useful compounds in combating inflammatory disease.