MAP Kinase Signal Transduction Pathway Research Articles

Exposure to toxic concentrations of aluminum activates a MAPK-like protein in cell suspension cultures of Coffea arabica

Addition of a toxic concentration of aluminum (Al) to cell suspension cultures of Coffea arabica L. induced the rapid and transient activation of a protein kinase that phosphorylates myelin basic protein (MBP), as revealed by in-gel kinase assays. This enzyme with an apparent molecular mass of 58 kDa was activated shortly after cells were exposed to 50 μM AlCl 3, a concentration previously shown to produce toxicity in plant cells in vitro. The activity of this kinase dropped to basal levels after 20 min of Al addition; this activity is specific for MBP as it could not be detected when casein or histone H1 were used as substrates. Analysis of the same cell extracts with antibodies that specifically recognize bis-phosphorylated (active) mitogen-activated protein kinases (MAP kinases), revealed the presence of a phosphoprotein with an apparent molecular mass of 58 kDa, which showed the same response to Al as the protein kinase revealed by the in-gel kinase assays. Furthermore, immunoprecipitation with an antibody directed against mammalian MAP kinases depleted both the enzymatic activity and the phosphoprotein from the cell extracts, suggesting that the 58 kDa kinase and the 58 kDa phosphoprotein from C. arabica cells are the same protein, and that it can be actually a member of the MAP kinase family of protein kinases. Since its activity is enhanced dramatically after addition of AlCl 3 to the medium, we can speculate that Al toxicity in plants could be perceived through the MAP kinase signal transduction pathway.

P42/p44 Mitogen‐Activated Protein Kinase Signal Transduction Pathway: A Novel Target for the Treatment of Hormone‐Resistant Prostate Cancer?

Prostate cancer is the second leading cause of cancer deaths in men. Conventional therapies produce a high rate of cure for patients with localized prostate cancer, but there is no cure once the disease has spread beyond the prostate. Androgen withdrawal remains the only treatment for these men with clinically advanced disease; however, most of these men, who initially respond to hormone ablation therapy, fail and the disease progresses. There is at present no effective treatment for hormone-independent prostate cancer. Several lines of evidence suggest a role of p42/p44 mitogen-activated protein kinase (p42/p44 MAP kinase) signal transduction pathways in prostate cancer. At the molecular level, a variety of genetic alterations lead to an epigenetic mechanism by which a feedback autocrine loop between membrane receptors and associated ligands serves as an essential component of the growth, proliferation, and metastasis of prostate cancer at an advanced and androgen-independent stage. Peptide growth factors are known to exert their effects by a complex array of mechanisms primarily mediated by the p42/p44 MAP kinase signal transduction pathway. Thus, we hypothesized that MAP kinase signal transduction pathways could serve as new and novel targets in prostate cancer therapy. In this article we provide an overview of the role played by MAP kinase signal transduction in the prostate.

P42/p44 Mitogen‐Activated Protein Kinase Signal Transduction Pathway Regulates Interleukin‐6 Expression in PC3 Cells, a Line of Hormone‐Refractory Prostate Cancer Cells

The mechanisms that regulate prostate cancer growth and proliferation are not fully understood. IL-6, a multifunctional cytokine, has been shown to play an important role in prostate cancer biology. Functional role of MAP-kinase signal transduction pathways in prostate biology has not been evaluated in detail. In the present study we evaluated the effects of modulation of p42/44 MAP kinase signal transduction pathway on IL-6 expression and secretion by PC3 cells, a line of hormone refractory prostate cancer cells. Results presented, herein, demonstrate that modulation of p42/44 MAP kinase activity results in partial inhibition of synthesis and secretion of IL-6. These data suggest that modulation of p42/p44 may result in regulation of other survival pathways as well.

Militarinone A induces differentiation in PC12 cells via MAP and Akt kinase signal transduction pathways

The fungal metabolite militarinone A (MILI A) promotes neurite outgrowth in PC12 cells. This study was conducted to investigate the signaling pathways involved in the cellular differentiation processes induced by the compound, with a focus on cascades implicated with nerve growth factor (NGF)-mediated neuritogenesis. MILI A possessed pronounced amphiphilic properties. The compound rapidly accumulated in the cell membrane and was slowly released into the cytoplasma. In primed PC12 cells, an early activation of protein kinase B (Akt), representing a downstream target of phosphoinositol 3 (PI 3) kinase, and a delayed phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), and of transcription factor cAMP responsive element binding protein (CREB) was found. The NGF-dependent activation of c-Jun amino terminal kinase (SAPK/JNK1) was potentiated. Morphological differentiation of cells and the phosphorylation of specific signal molecules were blocked by the MAP kinase (MEK1) inhibitor PD098059, the PI 3-kinase (PI 3K) inhibitor wortmannin and the adenylyl cyclase inhibitor 9-cyclopentyladenine.

Regulation of MAO-A and MAO-B gene expression.

MAO A and B genes are made of 15 exons with identical exon-intron organization. They are located on X-chromosome organized in opposite direction, tail to tail with 24 kb apart. Both promoters are GC-rich and regulated by transcription factor Sp1. However, they have distinctly different features. MAO B gene, but not MAO A gene, has TATA box. MAO B promoter contains two clusters of overlapping Sp1 sites, the CACCC repressor element. Transcription factors Sp1 and Sp4 can activate MAO B promoter activity through the proximal cluster of Sp1 sites and its activation can be repressed by the over-expression of Sp3 and a related family member, BTEB2. Decreased methylation and transcription repressor Sp3 upregulate human MAO B, but not MAO A, gene expression during Caco-2 differentiation. MAO B, but not MAO A gene, could be activated by PMA (phorbol 12-myristate 13-acetate) by protein kinase C, MAPkinase signal transduction pathway involves cJun and Egr-1. The differences in MAO A and B gene regulation may explain the different tissue-specific expression and functions of these two important isoenzymes.

The T1796A mutation of the BRAF gene is absent in Spitz nevi.

BRAF, a serine/threonine kinase, is a component of the retrovirus-associated sequence (RAS)-RAF-extracellular-regulated protein kinase (ERK)-MAP kinase signal transduction pathway mediating signals from RAS to ERK. The T1796A single point mutation in exon 15 of the BRAF gene has recently been reported in a high percentage of malignant melanomas and benign melanocytic lesions such as congenital nevi, compound nevi, intradermal nevi and dysplastic nevi. The T1796A mutation has been shown to promote cell proliferation. We screened 21 Spitz nevi and six spitzoid malignant melanomas for the presence of the T1796A BRAF mutation. The T1796A BRAF mutation could not be detected in any of the 21 Spitz nevi but was present in two of the six spitzoid malignant melanomas. Our results, in conjunction with data from a previous investigation, suggest that the melanocytic proliferation of Spitz nevi might be induced by components of the RAS-RAF-ERK-MAP kinase pathway different from BRAF, possibly combined with other genetic aberrations. The lack of the T1796A BRAF mutation might be of practical importance in distinguishing Spitz nevi from other melanocytic lesions simulating Spitz nevi as a part of a future complex diagnostic assay.

Diallyl disulfide-induced G2/M arrest of human gastric cancer MGC803 cells involves activation of p38 MAP kinase pathways.

To determine the role of p38 MAP kinase signal transduction pathways in diallyl disulfide (DADS)-induced G2/M arrest in human gastric cancer MGC803 cells. MGC803 cell growth inhibition was measured by MTT assay. Phase distribution of cell cycle was analyzed by flow cytometry. Expression of Cdc25C, p38, phosphorylation of p38 (pp38) were determined by Western blotting. MTT assay showed that SB203580, a specific p38 MAPK inhibitor blocked DADS-induced growth inhibition. Flow cytometry analysis revealed that treatment of MGC803 cells with 30 mg/L DADS increased the percentage of cells in the G2/M phase from 9.3% to 39.4% (P<0.05), whereas inhibition of p38 activity by SB203580 abolished induction of G2/M arrest by DADS. Western blotting showed that phosphorylation of p38 was increased 3.52-fold following treatment of MGC803 cells with 30 mg/L DADS for 20 min (P<0.05), whereas Cdc25C was decreased 68% following treatment of MGC803 cells with 30 mg/L DADS for 24 h (P<0.05). Decreased Cdc25C protein expression by DADS was attenuated by SB203580 (P<0.05). DADS-induced G2/M arrest of MGC803 cells involves activation of p38 MAP kinase pathways. Decreased Cdc25C protein expression by p38 MAPK played a crucial role in G2/M arrest after treatment with DADS.

Role of p38 MAP kinase signal transduction in apoptosis and survival of renal epithelial cells.

The p38 MAP kinase signal transduction pathway is activated by various forms of cellular stress. While, in many tissues, activation of p38 MAP kinase is associated with apoptosis, in some tissues p38 activation is critical for survival. Oxalate deposits are associated with several pathological conditions that involve aberrant proliferation and cellular apoptosis. Studies from our laboratory demonstrated that exposure of renal epithelial cells to oxalate and COM-crystals resulted in activation of the p38 MAP kinase pathway. Moreover, the inhibition of the p38 MAO kinase pathway resulted in the inhibition of oxalate as well as COM-crystal-induced reinitiation of the DNA synthesis. These results suggest a critical role for the p38 MAP kinase pathway in pathological conditions associated with cellular proliferation and apoptosis following deposition of calcium oxalate.

Involvement of MAP kinase signal transduction pathway in UVB-induced activation of macrophages in vitro

Ultraviolet B (UVB) radiation causes a variety of biological effects that can be either beneficial or harmful for human health. To exert these effects on a cellular basis, UVB induces a variety of signaling pathways, which act in an independent and additive ways. However, the UVB-induced signal transduction mechanism(s) till date remains not well understood. In the current investigation, we demonstrate p42/44, p38 and JNK MAPK activation in murine peritoneal macrophages on irradiation with UVB as determined by the expression of phospho-p42/44 MAPK, -p38 and -JNK. These responses were found to be rapid and time-dependent, detectable as early as 5min post UVB irradiation 50mJ/cm2 and reached a peak by 15min. UVB-induced phosphorylation of p42/44, p38 and JNK MAPKs was found to be blocked by Gαi-protein inhibitor, pertussis toxin; tyrosine kinase inhibitor, genistein; and PI3-K inhibitor, wortmannin. The activation of downstream transcription factors, c-jun and c-fos was also observed in response to UVB irradiation. The role of PD98059, SB202190, genistein, and wortmannin in regulating UVB-induced c-jun and c-fos activation and transcription was also investigated. The functional consequences of UVB-induced p42/44 MAPK and p38 MAPK activity were studied using TNF-α and NO production in the macrophages. The data suggests the involvement of p42/44, p38 and JNK MAPK pathway and subsequent induction of c-fos and c-jun in the signal transduction process leading to the activation of macrophages exposed to UVB.

MAP kinase phosphatase 1: a novel mediator of biological effects of glucocorticoids?

Synthetic glucocorticoids (GCs) potently inhibit the expression of pro-inflammatory genes and are widely used in the treatment of inflammatory diseases. However, some patients are resistant to the therapeutic effects of GCs, and many suffer deleterious side effects from these drugs. Furthermore, the precise mechanisms by which GCs inhibit pro-inflammatory gene expression remain unclear. A number of recent papers report that GCs induce the sustained expression of MAP kinase (MAPK) phosphatase 1 (MKP-1), a negative regulator of MAPK signal transduction pathways. The potential relevance of MKP-1 to some of the biological effects of GCs is discussed.

Phosphorylation of Hippocampal Erk-1/2, Elk-1, and p90-Rsk-1 during Contextual Fear Conditioning: Interactions between Erk-1/2 and Elk-1

The phosphorylation of proteins involved in the MAP kinase signal transduction pathway was investigated during associative learning of C57BL/6J mice. Context-dependent fear conditioning, consisting of a single exposure of mice to a context followed by foot shock, was employed as a learning paradigm. Control groups consisted of mice exposed to context only or an immediate shock in the context. Coincident up-regulation of phosphorylated Erk-1/2 and Elk-1 was observed in the CA3 hippocampal subfield and dentate gyrus 30 min after fear conditioning but not after the control paradigms. Phosphorylated Erk-1/2 and Elk-1 were associated and predominantly colocalized in the mossy fibers. In vitro kinase assays showed that hippocampal Erk-1/2 phosphorylates Elk-1. Notably, Elk-1 in turn enhances the phosphorylation of Erk-1/2 and its downstream target p90Rsk-1. Increased phosphorylation and nuclear translocation of p90Rsk-1 was also demonstrated in the CA3 hippocampal area in vivo during contextual fear conditioning. The observed interactions between hippocampal Elk-1 and Erk-1/2 proteins may affect the consolidation of contextual memories through activation of the downstream nuclear targets of Erk-1/2, such as p90Rsk-1, without requiring nuclear translocation of Elk-1 and Erk-1/2.

Distinct Signaling Pathways Are Activated in Response to Mechanical Stress Applied Axially and Transversely to Skeletal Muscle Fibers

In the diaphragm muscle we tested the hypothesis that MAP kinase signaling pathways are activated by mechanical stress and such signaling pathways are dependent on the direction in which mechanical stress is applied. Although equal magnitudes of mechanical stress were applied axially and transversely a greater level of activation of ERK1/2, p38, Raf-1, p90 RSK, Elk-1, and the DNA binding activity of AP-1 transcription factor was produced when the muscle was stretched transversely than when stretched axially. A significant up-regulation in protein tyrosine phosphorylation was observed in axially or transversely loaded diaphragm muscles and the activation of ERK1/2 was completely inhibited by genistein (protein-tyrosine kinase inhibitor). Pretreatment of muscles with wortmannin (phosphoinositide 3-kinase inhibitor), TMB-8 (antagonist of intracellular calcium release), GF109203X (PKC inhibitor), or PD98059 (MEK1/2 inhibitor) blocked the activation of ERK1/2 kinases in response to axial but not to transverse loading. On the other hand, pretreatment of muscles with protein kinase A inhibitors H-7 and KT5720 completely suppressed the activation of ERK1/2 in response to transverse loading only. Taken together with the alterations of MAP kinases and the findings of elevations of downstream transcription targets, our data are consistent with two distinct MAP kinase signal transduction pathways in response to mechanical stress.

Requirement of the JIP1 scaffold protein for stress-induced JNK activation.

The c-Jun N-terminal kinase (JNK) signal transduction pathway is activated in response to the exposure of cells to environmental stress. Components of the JNK signaling pathway interact with the JIP1 scaffold protein. JIP1 is located in the neurites of primary hippocampal neurons. However, in response to stress, JIP1 accumulates in the soma together with activated JNK and phosphorylated c-Jun. Disruption of the Jip1 gene in mice by homologous recombination prevented JNK activation caused by exposure to excitotoxic stress and anoxic stress in vivo and in vitro. These data show that the JIP1 scaffold protein is a critical component of a MAP-kinase signal transduction pathway.

MAP kinase signal transduction pathways in plants.

The mitogen-activated protein kinase (MAP kinase) signal transduction cascades are routes through which eukaryotic cells deliver extracellular messages to the cytosol and nucleus. These signalling pathways direct cell division, cellular differentiation, metabolism, and both biotic and abiotic stress responses. In plants, MAP kinases and the upstream components of the cascades are represented by multigene families, organized into different pathways which are stimulated and interact in complex ways. Experimental strategies for the analysis of MAP kinase cascades include the yeast two-hybrid system; using this approach in vitro interactions between specific MAP kinase cascade components have been analysed and putative plant cascades postulated. Transient transformation of protoplasts with epitope-tagged kinases has allowed cascades to be tested in planta. There is clear evidence for the involvement of MAP kinases in plant cell division and in the regulation of auxin signalling. Biotic (pathogens and pathogen-derived elicitors from fungi, bacteria and viruses) and abiotic stresses including wounding, mechanical stimulation, cold, drought and ozone can elicit defence responses in plants through MAP kinase pathways. There are data suggesting that ABA signalling utilizes a MAP kinase pathway, and probably ethylene and perhaps cytokinins do so also. The objective of this paper is to review this rapidly advancing field. Contents Summary 67 I. Introduction 68 II. Background 68 III. MAP kinase targets and targeting specificity 69 IV. Assays and inhibitors 70 V. Two well characterized MAP kinase pathways, Hog1 and Sevenless 71 VI. MAP kinases in plants 73 VII. MAP kinases and cell division 76 VIII. MAP kinases and plant hormones 76 IX. MAP kinase and abiotic stress 78 X. MAP kinase and biotic stress 80 XI. Future perspectives for MAP kinase research in plants 83 Acknowledgements 84 References 84.

Prevention of Cardiac Hypertrophy with Omega 3-Fatty Acids: Potential Cell Signaling Targets.

Epidemiological studies of Greenland Eskimos and Japanese have suggested that eating fish oil and marine animals can prevent heart disease. The beneficial effects of fish oils are attributed to their omega 3-fatty acid (O3FA) content, particularly, eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). DHA and EPA in the diet influence the fatty acid composition of plasma membrane phospholipids in cardiac tissues, which may affect cardiac cell functions. However, very little is known about the cellular and molecular mechanisms that mediate O3FA-induced cardio-protective effects. This review describes the potential cellular targets that can be modulated by O3FAs to regulate cardiac-related illnesses, particularly, cardiomyocyte hypertrophy. Among various biochemical derangements, the increase in intracellular Ca2+ ([Ca2+]i) allows the development of cardiac hypertrophy. Elevation of [Ca2+]i acts as a central intracellular signaling system by which hormones and growth factors regulate many different processes, such as secretion, metabolism, cell growth, differentiation, and cell contractility. Recent studies clearly suggest that O3FAs have profound effects on reducing [Ca2+]i levels by regulating both influx of Ca2+ through Ca2+ channels and mobilization of Ca2+ from intracellular stores. These fatty acids modulate Ca2+ current through the L-type calcium channels, and the effects occur within minutes of adding EPA or DHA to the medium. O3FAs can also regulate calcium mobilization from intracellular stores by affecting phosphatidylinositol cycle, phospholipase C activities, and inositol 1, 4, 5 trisphosphate generation. The effect of O3FAs on reducing the [Ca2+]i levels could be one of the mechanisms for preventing cardiac hypertrophy. In addition to affecting [Ca2+]i levels, these fatty acids can also affect other signaling pathways, including alterations in receptor affinity and density, activities of adenylate and guanylate cyclase, and cyclic nucleotide phosphodiesterase activities. There is no direct evidence that O3FAs affect src, ras, and MAP kinase signal transduction pathways in cardiac tissues, but in other cellular systems these pathways can be modulated by O3FAs. It therefore appears that a blockade of src, ras, and MAP kinase pathways, which is known to be involved in the development of cardiac hypertrophy, could be an effective target for O3FAs. Another important process in the development of cardiac hypertrophy is the activation of protein kinase C (PKC) isoenzymes. PKC activation leads to stimulation of specific pathways that mediate protein synthesis in cardiomyocytes. There is clear evidence that O3FAs affect the translocation and activation of PKC in cardiac tissues through multiple mechanisms. The modulation of PKC activities therefore could be a potent target in regulating cardiac hypertrophy and other cardiac-related abnormalities. In conclusion, these recent studies suggest that O3FAs could prevent the development of hormonal-induced cardiac hypertrophy by acting on multiple cellular signaling pathways.

Stimulation of Insulin-Like Growth Factor Binding Protein-1 Synthesis by Interleukin-1 : Requirement of the Mitogen-Activated Protein Kinase Pathway

Insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is a 28-kDa plasma protein that binds to IGF-I and IGF-II with high affinity. IGFBP-1 is elevated in the blood as a result of sepsis, AIDS, excessive alcohol consumption, and diabetes and may, in part, be responsible for the wasting observed during these pathophysiological conditions. The liver is the principal site of IGFBP-1 synthesis, and we have previously shown that proinflammatory cytokines can directly stimulate IGFBP-1 secretion in a human hepatoma cell line (HepG2). The purpose of the present study was to investigate the role of the MAP kinase pathway in regulating IGFBP-1 synthesis by IL-1β. We show that IL-1β stimulates the phosphorylation of ERK-1 and -2 in a time- and dose-dependent manner. In addition, the MAP kinase-kinase MEK-1 and the ribosomal S6-kinase RSK-1 are also phosphorylated in response to IL-1β. The transcription factor CREB, a potential substrate of both protein kinase A (PKA) and RSK-1, is phosphorylated in response to IL-1β and cAMP in HepG2 cells. The ability of IL-1β to stimulate the expression of IGFBP-1 and the phosphorylation of the above kinases was specifically inhibited by PD98059, a MEK-1 inhibitor. cAMP also stimulated IGFBP-1 synthesis, but PD98059 failed to block the cAMP effect. Conversely, a PKA inhibitor (H-89) inhibited the ability of cAMP, but not IL-1β to stimulate IGFBP-1 synthesis. The effect of IL-1β and cAMP on IGFBP-1 messenger RNA (mRNA) accumulation was additive. IL-1β, cAMP, PD98059, and H-89 had similar effects on the accumulation of IGFBP-1 protein and mRNA. IL-1β and cAMP did not change the half-life of IGFBP-1 mRNA, but PD98059 and SB202190, a p38 MAP kinase inhibitor, destabilized IGFBP-1 mRNA and blocked the phosphorylation of RSK-1 in response to IL-1β. Our data demonstrate that the MAP kinase signal transduction pathway plays an important role in the regulation of IGFBP-1 synthesis by IL-1β.

Stimulation of insulin-like growth factor binding protein-1 synthesis by interleukin-1beta: requirement of the mitogen-activated protein kinase pathway.

Insulin-like growth factor (IGF) binding protein-1 (IGFBP-1) is a 28-kDa plasma protein that binds to IGF-I and IGF-II with high affinity. IGFBP-1 is elevated in the blood as a result of sepsis, AIDS, excessive alcohol consumption, and diabetes and may, in part, be responsible for the wasting observed during these pathophysiological conditions. The liver is the principal site of IGFBP-1 synthesis, and we have previously shown that proinflammatory cytokines can directly stimulate IGFBP-1 secretion in a human hepatoma cell line (HepG2). The purpose of the present study was to investigate the role of the MAP kinase pathway in regulating IGFBP-1 synthesis by IL-1beta. We show that IL-1beta stimulates the phosphorylation of ERK-1 and -2 in a time- and dose-dependent manner. In addition, the MAP kinase-kinase MEK-1 and the ribosomal S6-kinase RSK-1 are also phosphorylated in response to IL-1beta. The transcription factor CREB, a potential substrate of both protein kinase A (PKA) and RSK-1, is phosphorylated in response to IL-1beta and cAMP in HepG2 cells. The ability of IL-1beta to stimulate the expression of IGFBP-1 and the phosphorylation of the above kinases was specifically inhibited by PD98059, a MEK-1 inhibitor. cAMP also stimulated IGFBP-1 synthesis, but PD98059 failed to block the cAMP effect. Conversely, a PKA inhibitor (H-89) inhibited the ability of cAMP, but not IL-1beta to stimulate IGFBP-1 synthesis. The effect of IL-1beta and cAMP on IGFBP-1 messenger RNA (mRNA) accumulation was additive. IL-1beta, cAMP, PD98059, and H-89 had similar effects on the accumulation of IGFBP-1 protein and mRNA. IL-1beta and cAMP did not change the half-life of IGFBP-1 mRNA, but PD98059 and SB202190, a p38 MAP kinase inhibitor, destabilized IGFBP-1 mRNA and blocked the phosphorylation of RSK-1 in response to IL-1beta. Our data demonstrate that the MAP kinase signal transduction pathway plays an important role in the regulation of IGFBP-1 synthesis by IL-1beta.

Mitogen-activated protein kinase Mkp1 of Pneumocystis carinii complements the slt2Delta defect in the cell integrity pathway of Saccharomyces cerevisiae.

Signal transduction pathways are important in the adaptive response of microbes to their environment. A Pneumocystis carinii extracellular signal-regulated protein kinase (MAPK) homologue, Mkp1, has been isolated by sequence similarity screening of P. carinii genomic DNA. The Mkp1 of P. carinii shows closest homology to other fungal MAP kinases involved in cell integrity signal transduction cascades, including Slt2p/Mpk1p of Saccharomyces cerevisiae, Mkc1 of Candida albicans and Mps1 of Magnaporthe grisea. Defects of Slt2p in S. cerevisiae result in phenotypes of slow growth, and temperature sensitivity in the absence of an osmostabilizer. Overexpression of mkp1 in a strain with the slt2Delta defect fully restored the normal growth rate, and partially reduced lysis at elevated temperatures. Complementation of the slt2Delta defect by Mkp1 demonstrates that Mkp1 is a functional MAP kinase, and that it may be the MAP kinase component of a similar signal transduction cascade within P. carinii. Furthermore, Mkp1 is activated in vitro upon the exposure of P. carinii to conditions of oxidative stress. The investigation of a MAP kinase signal transduction pathway of P. carinii will result in both a better understanding of the mechanism the organism utilizes to respond to environmental changes, and a system to assay responses to these changes.

A MAP kinase encoded by the ubc3 gene of Ustilago maydis is required for filamentous growth and full virulence.

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this, we have taken a forward genetic approach. Previously, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cAMP to grow in the budding morphology. Complementation of one of these suppressor mutants led to the identification of ubc3, which is required for filamentous growth and encodes a MAP kinase most similar to those of the yeast pheromone response pathway. In addition to filamentous growth, the ubc3 gene is required for pheromone response and for full virulence. Mutations in the earlier identified fuz7 MAP kinase kinase also suppress the filamentous phenotype of the uac1 disruption mutant, adding evidence that both ubc3 and fuz7 are members of this same MAP kinase cascade. These results support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.

Bryostatin 1 enhances paclitaxel-induced mitochondrial dysfunction and apoptosis in human leukemia cells (U937) ectopically expressing Bcl-xL.

The effects of the protein kinase C (PKC) activator and down-regulator bryostatin 1 were examined with respect to paclitaxel-induced apoptosis and antiproliferative activity in human myeloid leukemia cells (U937) displaying enforced expression of the anti-apoptotic protein Bcl-xL. Overexpression of Bcl-xL blocked various aspects of paclitaxel-mediated apoptosis, including caspase-3 activation, degradation of poly(ADP-ribose) polymerase (PARP), loss of mitochondrial membrane potential (Delta Psim), and release of cytochrome c. However, subsequent (but not prior) exposure of paclitaxel-treated U937/Bcl-xL cells (500 nM; 6 h) to bryostatin 1 (10 nM; 15 h) restored the extent of apoptosis, caspase activation, and mitochondrial damage to levels approximating those in paclitaxel-treated empty-vector control cells (U937/Neo). Potentiation of paclitaxel-induced apoptosis by bryostatin 1 in U937/Bcl-xL cells occurred primarily in the G2M cell population, and was associated with alterations in Bcl-xL gel mobility and a reduction in paclitaxel-mediated stimulation of CDK1 activity. Enhancement of paclitaxel-induced apoptosis by bryostatin 1 in Bcl-xL overexpressors was accompanied by a corresponding reduction in clonogenic potential. In contrast to its effects on apoptosis, bryostatin 1 failed to restore paclitaxel-mediated increases in free Bax levels in U937/Bcl-xL cells. Lastly, the actions of bryostatin 1 were mimicked by a pharmacologic inhibitor of the MEK1/MAP kinase pathway (PD98059), but not by SB203580, an inhibitor of p 38 MAP kinase. Moreover, sequential exposure of both U937/Neo or/Bcl-xL cells to paclitaxel followed by bryostatin 1 or PD98059 was associated with a net reduction in MAP kinase activity. Collectively, these findings indicate that protection against paclitaxel-mediated mitochondrial dysfunction and apoptosis in human U937 leukemia cells conferred by Bcl-xL overexpression can be substantially overcome by bryostatin 1 and possibly other agents that interrupt the MAP kinase signal transduction pathway.