Extracellular Receptor Kinase Research Articles

Glutamate regulation of DARPP-32 phosphorylation in neostriatal neurons involves activation of multiple signaling cascades

Dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) plays a central role in medium spiny neurons in the neostriatum in the integration of various neurotransmitter signaling pathways. In its Thr-34-phosphorylated form, it acts as a potent protein phosphatase-1 inhibitor, and, in its Thr-75-phosphorylated form, it acts as a cAMP-dependent kinase inhibitor. Here, we investigated glutamate-dependent signaling cascades in mouse neostriatal slices by analyzing the phosphorylation of DARPP-32 at Thr-34 and Thr-75. Treatment with glutamate (5 mM) caused a complex change in DARPP-32 Thr-34 phosphorylation. An initial rapid increase in Thr-34 phosphorylation was NMDA/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/metabotropic glutamate-5 receptor-dependent and was mediated through activation of a neuronal nitric oxide synthase/nitric oxide/cGMP/cGMP-dependent kinase signaling cascade. A subsequent decrease in phosphorylation was attributable to activation of an NMDA/AMPA receptor/Ca2+/protein phosphatase-2B signaling cascade. This decrease was followed by rephosphorylation via a pathway involving metabotropic glutamate-5 receptor/phospholipase C and extracellular receptor kinase signaling cascade. Treatment with glutamate initially decreased Thr-75 phosphorylation through activation of NMDA/AMPA receptor/Ca2+/protein phosphatase-2A signaling. Thereafter, glutamate slowly increased Thr-75 phosphorylation through activation of metabotropic glutamate-1 receptor/phospholipase C signaling. Our analysis of DARPP-32 phosphorylation in the neostriatum revealed that glutamate activates at least five different signaling cascades with different time dependencies, resulting in complex regulation of protein kinase and protein phosphatase activities.

Imatinib-Induced Regression of AIDS-Related Kaposi's Sarcoma

Activation of the platelet-derived growth factor (PDGF) and c-kit receptors has been proposed as important in mediating the growth of AIDS-related Kaposi's sarcoma (KS). We investigated the response of KS to the PDGF receptor (PDGFR)/c-kit inhibitor, imatinib mesylate, and investigated the effect of this therapy on critical signal transduction intermediates. Ten male patients with AIDS-related cutaneous KS, which progressed despite chemotherapy and/or highly active antiretroviral therapy, received imatinib mesylate administered orally, 300 mg twice daily. Clinical response was determined by serial tumor measurements. To determine biologic and histologic response, skin lesion biopsies were obtained at baseline and following 4 weeks of therapy. Five of 10 participants had a partial response by tumor measurements. Biopsies after 4 weeks of therapy demonstrated histologic regression in four of six patients. Four patients' tumor biopsies were assessable for immunohistochemistry end points pre- and post-therapy. These demonstrated inhibition of PDGFR and its downstream effector, extracellular receptor kinase, which is a member of the mitogen-activated protein kinase family. The most common adverse event was diarrhea, which led to dose reduction in six patients. Imatinib mesylate administered orally twice daily for AIDS-related KS results in clinical and histologic regression of cutaneous KS lesions within 4 weeks. These promising results demonstrate that inhibition of the c-kit and/or PDGF receptors may represent an effective strategy for treating KS.

Differences in Cell Activation byChlamydophila pneumoniaeandChlamydia trachomatisInfection in Human Endothelial Cells

Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linked Chlamydophila pneumoniae infection to the development of chronic vascular lesions and coronary heart disease. In this study, we characterized C. pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH2 kinase (JNK). Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway. Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression. While Chlamydia trachomatis was also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1. These effects were specific for a direct stimulation with viable C. pneumoniae and independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes. Thus, C. pneumoniae triggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis.

Extracellular receptor kinase and cAMP response element binding protein activation in the neonatal rat heart after perinatal cocaine exposure.

Prenatal exposure to cocaine has been shown to induce an increase in the myocardial expression and activation of the cAMP response binding protein (CREB), a transcriptional factor that has been shown to regulate gene expression. Several different kinases, including protein kinase A, calcium calmodulin kinase II, and mitogen-activated protein kinase can induce phosphorylation of CREB at serine 133, a necessary step for CREB activation. We examined whether the mitogen-activated protein kinase-extracellular receptor kinase (ERK) pathway may be involved in mediating the serine 133 CREB phosphorylation in cardiac nuclei after perinatal cocaine exposure. Pregnant rats were treated daily with saline or cocaine at 60 mg/kg (C60) by intragastric administration during the entire gestational period, and treatment was continued in the nursing dams after delivery until the time of the study. Nuclear extracts were isolated from hearts of 1-d- and 7-d-old neonatal rats. We performed immunoblotting experiments using an antibody that recognized CREB with phosphorylation specifically at the serine 133 site and an antibody that recognized both the phosphorylated and the unphosphorylated forms of CREB, as well as antibodies for total ERK, phospho-ERK, total ribosomal S6 kinase 1 (RSK1), RSK2, and phospho-RSK. We assessed the interaction of RSK with CREB or CREB-binding protein by performing co-immunoprecipitation experiments. We found that perinatal cocaine exposure increased both phospho-ERK and phospho-RSK expression, indicative of an increased activity of these two enzymes. Furthermore, we demonstrated that phospho-RSK was immunoprecipitated with CREB in all neonatal cardiac nuclei and that the greatest interaction was found in day 7 hearts after perinatal cocaine exposure. Our results thus illustrate that the ERK-RSK pathway was active in the postnatal rat heart at 1 and 7 d of age and that this pathway may mediate the increase in myocardial CREB activation after perinatal cocaine exposure in the day 7 hearts.

Novel aspects in regulated expression of the renal type IIa Na/Pi-cotransporter

Proximal tubular phosphate (P(i)) reabsorption is a key element in overall phosphate homeostasis; physiologic/pathophysiologic alterations are related to the control of brush border membrane expression (regulated endocytosis) of the type IIa sodium (Na)/phosphate(P(i))-cotransporter (NaPi-IIa). The carboxy terminus of NaPi-IIa contains sequences important for its apical delivery/expression; the last three amino acids are involved in PSD95/DglA/ZO-1 (PDZ) interactions involving NaPi-IIa, Na/H exchanger-regulatory factor 1 (NHERF1/2), and PDZK1/2 (apical scaffold). Regulated endocytosis of NaPi-IIa [e.g., parathyroid hormone (PTH)-induced] is reduced in megalin-deficient mice; internalization occurs via clathrin-coated structures, early endosomes, and finally leads to lysosomal degradation. NaPi-IIa contains, in the third intracellular loop, a sequence motif required for internalization. Different hormonal [e.g., PTH, atrial natriuretic peptide (ANP), also nitric oxide (NO)] and nonhormonal factors activate a variety of intracellular signaling cascades [protein kinase A (PK-A), protein kinase C (PK-C), protein kinase G (PK-G), extracellular receptor kinase (ERK)-1/2] leading (by unknown mechanisms) to NaPi-IIa internalization. Different phosphatonins [e.g., fibroblast growth factor (FGF)-23, frizzled related protein (FRP)-4, matrix extracellularphosphoglycoprotein (MEPE)], associated with different pathophysiologic states of renal P(i)-handling, seem also to control apical expression of NaPi-IIa. Internalization of NaPi-IIa first requires its removal from the apical scaffold. This scaffold can also be considered as a regulatory scaffold containing also protein kinase A (PK-A)-anchoring proteins (AKAPs, ezrin) and the apical PTH receptor. The role of the different components of the regulatory scaffold in regulated endocytosis of NaPi-IIa is at present unknown.

Modulation of Hippocampal Plasticity and Cognitive Behavior by Short-term Blueberry Supplementation in Aged Rats

During aging, reductions in hippocampal neurogenesis are associated with memory decline indicating a causal relationship. Indeed, insulin-like growth factor-1 (IGF-1), a major activator of the extracellular receptor kinase pathway that is central in learning and memory processes, is also a key modulator of hippocampal neurogenesis. Previously, we showed that age-related declines in spatial memory tasks can be improved by antioxidant-rich diets containing blueberries. In this study, to begin to understand the mechanisms responsible for the beneficial effects of blueberries, we assessed changes in hippocampal plasticity parameters such as hippocampal neurogenesis, extracellular receptor kinase activation, and IGF-1 and IGF-1R levels in blueberry-supplemented aged animals. Our results show that all these parameters of hippocampal neuronal plasticity are increased in supplemented animals and aspects such as proliferation, extracellular receptor kinase activation and IGF-1 and IGF-1R levels correlate with improvements in spatial memory. Therefore, cognitive improvements afforded by polyphenolic-rich fruits such as blueberries appear, in part, to be mediated by their effects on hippocampal plasticity.

Clustering of cellular prion protein induces ERK1/2 and stathmin phosphorylation in GT1-7 neuronal cells

The physiological role of the prion protein is largely unknown. Here, clustering of prion at the surface of GT1-7 cells was observed upon anti-prion antibody treatments. This clustering was associated with a rapid and transient phosphorylation of the mitogen activated protein kinases (MAPKs) extracellular receptor kinases 1 and 2 (ERK1/2), and also of the microtubule-destabilizing protein stathmin at serine 16. The specificity of this antibody-mediated activation was ascertained by its inhibition by prion small interfering RNA. The phosphorylation of ERK1/2 but not that of stathmin was abolished by the MAPK/ERK kinase 1 inhibitor U0126, whereas both signaling pathways were blocked by the specific inhibitor of the epidermal growth factor receptor AG1478, suggesting the likely recruitment of this receptor upon prion clustering.

BMP4 supports self-renewal of embryonic stem cells by inhibiting mitogen-activated protein kinase pathways.

The fate of pluripotent stem cells is tightly controlled during early embryonic development. Both the derivation and the maintenance of embryonic stem cells (ES cells) in vitro depend on feeder cell-derived growth factors that are largely unidentified. To dissect the mechanisms governing pluripotency, we conducted a screen to identify factors that are produced by mouse embryonic fibroblast STO cells and are required to maintain the pluripotency of ES cells. One of the factors is bone morphogenetic protein 4 (BMP4). Unexpectedly, the major effect of BMP4 on the self-renewal of ES cells is accomplished by means of the inhibition of both extracellular receptor kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways, and inhibitors of ERK and p38 MAPKs mimic the effect of BMP4 on ES cells. Importantly, inhibition of the p38 MAPK pathway by SB203580 overcomes the block in deriving ES cells from blastocysts lacking a functional Alk3, the BMP type IA receptor. These results uncover a paradigm for BMP signaling in the biology of pluripotent stem cells.

Cell signaling by imidazoline-1 receptor candidate, IRAS, and the nischarin homologue.

IRAS transfection into Chinese hamster ovary (CHO) or pheochromocytoma (PC-12) cell lines leads to the appearance of nonadrenergic binding sites for radiolabeled-clonidine. Nischarin is the mouse homologue of IRAS. IRAS seems to be a cytosolic protein that is anchored to the intracellular side of plasma membranes by a POX domain. Previous studies of IRAS-transfected HEK293 cells, and Nischarin-transfected 3T3 cells have shown this protein can intrinsically mediate cell growth and differentiation independent of imidazoline drugs through binding to insulin receptor substrates (HEK293 cells) and fibronectin receptors (3T3 cells). Herein, a growth-arrested PC-12 cell line stably transfected with IRAS is shown to express lower basal and nerve growth factor-stimulated levels of the activated form of extracellular receptor kinase than found in a vector-only transfected control cell line treated similarly. These findings suggest that IRAS is a membrane-associated mediator of receptor signaling.

Protein kinase-A activity in PRKAR1A-mutant cells, and regulation of mitogen-activated protein kinases ERK1/2.

Carney complex (CNC) is caused by PRKAR1A-inactivating mutations. PRKAR1A encodes the regulatory subunit type I-alpha (RIalpha) of the cAMP-dependent kinase (PKA) holoenzyme; how RIalpha insufficiency leads to tumorigenesis remains unclear. In many cells PKA inhibits the extracellular receptor kinase (ERK1/2) cascade of the mitogen-activated protein kinase (MAPK) pathway leading to inhibition of cell proliferation. We investigated whether the PKA-mediated inhibitory effect on ERK1/2 is affected in CNC cells that carry germline PRKAR1A mutations. PKA activity both at baseline and after stimulation with cAMP was augmented in cells carrying mutations. Quantitative message analysis showed that the main PKA subunits expressed were type I (RIalpha and RIbeta) but RIalpha was decreased in mutant cells. Immunoblot assays of ERK1/2 phosphorylation by the cell- and pathway-specific stimulant lysophosphatidic acid (LPA) showed activation of this pathway in a time- and concentration-dependent manner that was prevented by a specific inhibitor. There was a greater rate of growth in mutant cells; forskolin and isoproterenol inhibited LPA-induced ERK1/2 phosphorylation in normal but not in mutant cells. Forskolin inhibited LPA-induced cell proliferation and metabolism in normal cells, but stimulated these parameters in mutant cells. These data were also replicated in a pituitary tumor cell line carrying the most common PRKAR1A mutation (c.578del TG), and an in vitro construct of mutant PRKAR1A that was recently shown to lead to augmented PKA-mediated phosphorylation. We conclude that PKA activity in CNC cells is increased and that its stimulation by forskolin or isoproterenol increases LPA-induced ERK1/2 phosphorylation, cell metabolism and proliferation. Reversal of PKA-mediated inhibition of this MAPK pathway in CNC cells may contribute to tumorigenesis in this condition.

Growth factor-dependent activation of the MAPK pathway in human pancreatic cancer: MEK/ERK and p38 MAP kinase interaction in uPA synthesis.

Increased expression of the hepatocyte growth factor (HGF) receptor (c-met) and urokinase type plasminogen (uPA) correlated with the development and metastasis of cancers. To investigate the role of HGF/c-met signaling on metastasis in cancer cells stimulated with HGF, we examined the effects of a specific MEK1 inhibitor (PD98059) and a p38 MAP kinase inhibitor (SB203580) on HGF-induced uPA expression in pancreatic cancer cell lines, L3.6PL and IMIM-PC2. Pretreatment of PD98059 decreased HGF-mediated phosphorylation of extracellular receptor kinase (ERK), uPA secretion and expression of matrix metalloproteinases (MMP-2 and MMP-9) in a dose-dependent manner. In contrast, SB203580 pretreatment increased HGF-stimulated ERK phosphorylation, uPA secretion and expression of MMPs. SB203580 also reversed the inhibition of HGF-mediated ERK activation and uPA secretion in the PD98059-pretreated cells. These results suggest that ERK activation by HGF might play important roles in the metastasis of pancreatic cancer and the p38 MAPK pathway also involved in the HGF-mediated uPA secretion and metastasis by regulation of ERK pathway.

PI3 Kinase and not p42/p44 Appears to be Implicated in the Protection Conferred by Ischemic Preconditioning

M. M. Mocanu, R. M. Bell and D. M. Yellon. PI3 Kinase and not p42/p44 Appears to be Implicated in the Protection Conferred by Ischemic Preconditioning. Journal of Molecular and Cellular Cardiology (2002) 34, 661–668. Ischemic preconditioning results in an immediate phase of protection against lethal ischemia/reperfusion injury that is comprised of both irreversible necrosis and programmed cell death, apoptosis. We hypothesized that preconditioning may activate putative anti-apoptotic pathways, through the induction of either phosphatidyl inositol 3-OH kinase (PI3 kinase) or p42/p44 extracellular receptor kinase, attenuating total cell death. Isolated perfused rat hearts were preconditioned with two cycles of 5min ischemia and 10min reperfusion. Then they were frozen for Western blot analysis or subjected to 35min regional ischemia and 120min reperfusion prior to infarct size assessment. Selective PI3 kinase inhibitors, wortmannin (W, 100n M) and LY294002 (LY, 15μM) and the p42/p44 inhibitor, PD98059 (PD,10and 50μM), were individually infused during the preconditioning protocol. One further group of hearts received both inhibitors (W and PD). The results were expressed as percentage of infarction within the risk zone. Inhibition of PI3 kinase by either W or LYpartially abrogated the infarct sparing effect of ischemic preconditioning (I/R%: 44.6±2.7 in C, 17.6±2.0 in IP, vs 32.2±4.2 in W, and 30.9±2.6 in LY, P<0.05). Inhibition of ERK phosphorylation however, had no significant effect upon infarct size reduction (17.6±2.0 in ischemic preconditioning vs 21.4±3.0 in IP+10μM PD and 15.2±1.4 in IP+50μM PD, P>0.05). Western blot analysis confirmed that PD abrogated the phosphorylation of p42/p44 and LY the phosphorylation of AKT. Combined inhibition with PD+W failed to further attenuate protection (27.6±1.3%,P >0.1). These data appear to demonstrate that the PI3 kinase, but not the p42/p44 cascade, is implicated in early ischemic preconditioning.

Mitochondrial ROS initiate phosphorylation of p38 MAP kinase during hypoxia in cardiomyocytes.

The p38 mitogen-activated protein kinase (MAPK) is phosphorylated in response to oxidative stress. Mitochondria in cardiomyocytes increase their generation of reactive oxygen species (ROS) during hypoxia (1-5% O(2)). These ROS participate in signal transduction pathways involved in adaptive responses, including ischemic preconditioning and gene transcription. The present study therefore tested the hypothesis that hypoxia induces p38 MAPK phosphorylation by augmenting mitochondrial ROS generation. In cardiomyocytes, phosphorylation of p38 was observed in a PO(2)-dependent manner during hypoxia. This response was inhibited by rotenone, thenoyltrifluoroacetone, and myxothiazol, inhibitors of mitochondrial complexes I, II, and III, respectively. A similar inhibition was observed in the cells pretreated with anion channel inhibitor DIDS, which may block ROS release from mitochondria. During normoxia, increases in mitochondrial ROS elicited by azide (1-2 mM) or by the mitochondrial inhibitor antimycin A caused increased phosphorylation of p38. Brief treatment with exogenous H(2)O(2) during normoxia also induced phosphorylation of p38 as hypoxia, but this effect was not abolished by myxothiazol or DIDS. The antioxidant N-acetyl-cysteine abolished the p38 response to hypoxia, presumably by scavenging H(2)O(2), but the mitogen extracellular receptor kinase inhibitor PD-98059 did not inhibit p38 phosphorylation during hypoxia. Thus physiological hypoxia leads to p38 phosphorylation through a mechanism that requires electron flux in the proximal region of the mitochondrial electron transport chain, which suggests that either H(2)O(2) or superoxide participates in activating that process.

Determination of the epigenetic effects of ochratoxin in a human kidney and a rat liver epithelial cell line

Epidemiological studies have implicated ochratoxin A (OTA), a fungal metabolic-contaminant of animal and human food sources, in Balkan Endemic Nephropathy and renal tumors. Many environmental toxicants operate through nongenotoxic mechanisms that epigenetically control gene expression leading to a diseased state. Gap junctional intercellular communication (GJIC) plays a central role in the epigenetic control of genes in which alteration of normal GJIC has been implicated in many human pathologies, including cancer, teratogenesis, reproductive dysfunction and peripheral neuropathies. The cell proliferative stages of human diseases, such as cancer, also involves the induction of signal transduction pathways controlling the mitogenic steps, in which the mitogen activated protein kinases (MAPK), such as extracellular receptor kinase (ERK) and p38, are central to mitogenesis. We therefore determined the effects of OTA on GJIC and MAPK in a human kidney and rat liver epithelial cell line. OTA reversibly inhibited GJIC at noncytotoxic doses in the rat liver but not the human kidney cell line. Similarly, OTA was also a strong activator of MAPK, ERK and p38, in the rat liver cells but only weakly activated ERK and had no affect on p38 in the human kidney cell line. Another hallmark of human diseases is an abnormal alteration of apoptosis, also known as programmed cell death. We used our myc-transfected cell line, which exhibits higher levels of apoptosis, to test the effects of OTA on apoptosis. OTA greatly induced apoptosis in this cell line, which is contrary to the effects of most tumor promoters. In summary, OTA exhibits tumor promoting properties in the liver, but the effects of OTA on the human kidney epithelial cells suggested a lack of tumorigenic activity assuming that these epithelial cells, like the rat liver epithelial cells, are a primary target for carcinogens. These results also indicate that the nephrotoxicity of OTA either does not involve GJIC, assuming these epithelial cells play a vital role in kidney physiology, or that a more differentiated kidney cell type is the target for OTA toxicity, of which the role of GJIC remains unknown.

Lipopolysaccharide and CpG DNA synergize for tumor necrosis factor-alpha production through activation of NF-kappaB.

Unmethylated CpG motifs in bacterial DNA (CpG DNA) activate host innate immune responses synergistically with some other microbial products, such as endotoxins, and may contribute to disease pathogenesis through excessive production of proinflammatory cytokines. Because monocyte-derived tumor necrosis factor (TNF)-alpha is an important mediator of disease, we investigated whether CpG DNA and lipopolysaccharide (LPS) synergize for inducing TNF-alpha biosynthesis. CpG DNA and LPS synergistically induce TNF-alpha production in RAW264.7 cells and J774 cells through activation of NF-kappaB. Furthermore, transient transfection with a super-repressive mutant of IkappaBalpha (IkappaBalpha-AA) demonstrated that NF-kappaB plays a critical role in CpG DNA-mediated TNF-alpha expression. Like NF-kappaB activation, CpG DNA-induced activation of mitogen-activated protein kinases (MAPK) regulates TNF-alpha production. Both extracellular receptor kinase (ERK) and p38 can regulate TNF-alpha gene transcription induced by CpG DNA. Although CpG DNA at the higher concentration slightly enhanced LPS-mediated phosphorylation of ERK, it did not alter the LPS-mediated activation of c-Jun N-terminal kinase and p38. In addition, CpG DNA showed little or no enhancement of LPS-mediated AP-1 activation. These results suggest that CpG DNA- and LPS-mediated signals converge at or above the level of NF-kappaB and ERK, and that there are distinct, as well as common, signaling pathways which are utilized by both CpG DNA and LPS for activating various transcription factors and MAPK.

Neurotrophic Signaling in Normal and Degenerating Rodent Retinas

Several types of insult cause up-regulation of neurotrophic factors and their receptors in the retina resulting in decreased photoreceptor cell death from subsequent injury. This phenomenon is more prominent in rats than in mice and neurotrophic factors are more efficacious in rats than mice. If up-regulation of neurotrophic factor receptors on photoreceptor cells early in the course of degenerations contributes to neurotrophic factor survival-promoting activity, it may also increase the ability to detect neurotrophic factor-induced signaling in photoreceptors, particularly in rats. In this study, these hypotheses were investigated by performing immunohistochemical staining for the phosphorylated form of extracellular receptor kinase (pERK) or c-fos after intravitreous injection of neurotrophic factors in wild type rats or mice, or those with inherited retinal degenerations. In both rats and mice either early or late in the course of degeneration, or in wild type animals, intravitreous injection of brain-derived neurotrophic factor, ciliary neurotrophic factor, or fibroblast growth factor-2 caused immunostaining for pERK and c-fos in cells of the inner retina, particularly Müller cells, but not in photoreceptors. These data add to the mounting evidence suggesting that neurotrophic factors act indirectly through Müller cells to promote photoreceptor survival.

Rac/Cdc42 and p65PAK Regulate the Microtubule-destabilizing Protein Stathmin through Phosphorylation at Serine 16

We have identified a rapid protein phosphorylation event at residue serine 16 of stathmin using two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization mass spectrometry in combination with post-source decay analysis, which is induced by the epidermal growth factor receptor. Phosphorylation is specifically mediated by the small GTPases Rac and Cdc42 and their common downstream target, the serine/threonine kinase p65PAK. Both GTPases have previously been shown to regulate the dynamics of actin polymerization. Because stathmin destabilizes microtubules, and this process is inhibited by phosphorylation at residue 16, Rac and Cdc42 can potentially regulate both F-actin and microtubule dynamics.

Glutathione S-transferase p elicits protection against H2O2-induced cell death via coordinated regulation of stress kinases.

To elucidate mechanisms underlying glutathione S-transferase p (GSTp)-mediated cellular protection against oxidative stress-induced cell death, the effect of GSTp on stress signaling pathways was investigated before and after H2O2 treatment. Under nonstressed conditions, increased expression of GSTp via a tet-off-inducible GSTp in NIH 3T3 cells increased the phosphorylation of mitogen-activated protein (MAP) kinase kinase 4, p38, extracellular receptor kinase (ERK), and inhibitor of kappa-kinase (IKK), and reduced phosphorylation of MAP kinase kinase 7 and Jun NH2-terminal kinase (JNK). Whereas H2O2 treatment of cells induced JNK, p38, and IKK activities, in the presence of H2O2 and elevated GSTp expression there was an additional increase in ERK, p38, and IKK activities and a decrease in JNK activity. GSTp-mediated protection from H2O2-induced death was attenuated upon inhibition of p38, nuclear factor KB, or MAP kinase by dominant negative or pharmacological inhibitors. Conversely, expression of a dominant negative JNK protected cells from H2O2-mediated death. These data suggest that the coordinated regulation of stress kinases by GSTp, as reflected by increased p38, ERK, and nuclear factor kappaB activities together with suppression of JNK signaling, contributes to protection of cells against reactive oxygen species-mediated death.

Mechanism and function of a newly identified CpG DNA motif in human primary B cells.

The vertebrate immune system recognizes bacterial DNA based on the presence of unmethylated CpG-dinucleotides in particular base contexts ("CpG motifs"). In contrast to mice, knowledge about CpG-mediated effects on human B cells is poor. In the present study we identify and determine an optimal human CpG motif. A phosphodiester oligonucleotide containing this motif strongly stimulated CD86, CD40, CD54, and MHC class II expression, IL-6 synthesis, and proliferation of primary human B cells. These effects required internalization of the oligonucleotide and endosomal maturation. The molecular mechanism of action of this CpG motif was associated with the sustained induction of the NF-kappaB p50/p65 heterodimer and of the transcription-factor complex AP-1. Transcription-factor activation by CpG DNA was preceded by increased phosphorylation of the stress kinases c-Jun N-terminal kinase and p38, and of activating transcription factor-2. In contrast to CpG, signaling through the B cell receptor led to activation of extracellular receptor kinase and to phosphorylation of a different isoform of c-Jun N-terminal kinase. These studies define the structure of a highly active human CpG motif and characterize its molecular mechanism of action in primary human B cells.

Activation of neuronal extracellular receptor kinase (ERK) in Alzheimer disease links oxidative stress to abnormal phosphorylation.

Responses to increased oxidative stress may be the common mechanism responsible for the varied cytopathology of Alzheimer disease (AD). A possible link in support of this hypothesis is that one of the most striking features of AD, the abnormal accumulation of highly phosphorylated tau and neurofilament proteins, may be brought about by extracellular receptor kinase (ERK) whose activation is a common response to oxidative stress. In this study, we demonstrate that activated ERK is specifically increased in the same vulnerable neurons in AD that are the site of oxidative damage and abnormal phosphorylation. These findings suggest that ERK dysregulation, likley resulting from oxidative stress, could play an important role in the increased phosphorylation of cytoskeletal proteins observed in AD.