Nerve Growth Factor-mediated Activation of the Mitogen-activated Protein (MAP) Kinase Cascade Involves a Signaling Complex Containing B-Raf and HSP90

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway.

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The mitogen-activated protein kinase cascade is activated by B-Raf in response to nerve growth factor through interaction with p21ras.

Molecular and Cellular Biology ◽

10.1128/mcb.14.10.6944 ◽

1994 ◽

Vol 14 (10) ◽

pp. 6944-6953 ◽

Cited By ~ 103

Author(s):

R K Jaiswal ◽

S A Moodie ◽

A Wolfman ◽

G E Landreth

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Map Kinase ◽

Pc12 Cells ◽

Mitogen Activated Protein Kinase ◽

Stable Complex ◽

Nerve Growth ◽

Map Kinase Cascade ◽

Mitogen Activated Protein ◽

Kinase Cascade

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21ras-dependent signal transduction pathway in PC12 cells. The linkage between p21ras and MEK1 was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Raf1; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Raf1-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent M(r) of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Raf1, directly interacted with activated p21ras immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Raf1 to bind to activated p21ras. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p21ras following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Raf1 activity was not found to function within this pathway.

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Sustained activation of the mitogen-activated protein (MAP) kinase cascade may be required for differentiation of PC12 cells. Comparison of the effects of nerve growth factor and epidermal growth factor

Biochemical Journal ◽

10.1042/bj2880351 ◽

1992 ◽

Vol 288 (2) ◽

pp. 351-355 ◽

Cited By ~ 637

Author(s):

S Traverse ◽

N Gomez ◽

H Paterson ◽

C Marshall ◽

P Cohen

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Map Kinase ◽

Pc12 Cells ◽

Map Kinase Cascade ◽

Mitogen Activated Protein ◽

Kinase Cascade ◽

Epidermal Growth ◽

Sustained Activation

Stimulation of PC12 cells with nerve growth factor (NGF) increased mitogen-activated protein kinase kinase (MAPKK) activity > 20-fold after 5 min to a level that was largely sustained for at least 90 min. MAPKK activity was stimulated to a similar level by epidermal growth factor (EGF), but peaked at 2 min, declining thereafter and returning to basal levels after 60-90 min. Activation of MAPKK by either growth factor occurred prior to the activation of MAP kinase, consistent with MAPKK being the physiological activator of MAP kinase. The results demonstrate that the transient activation of MAPKK by EGF and its sustained activation by NGF underlies the transient and sustained activation of MAP kinase induced by EGF and NGF respectively. NGF or EGF induced the same two forms of MAPKK that were resolved on a Mono Q column. The Peak-1 MAPKK was activated initially and partially converted into the more acidic peak-2 MAPKK after prolonged growth-factor stimulation. The Peak-2 MAPKK was 20-fold more sensitive to inactivation by the catalytic subunit of protein phosphatase 2A. Stimulation with NGF caused a striking translocation of MAP kinase from the cytosol to the nucleus after 30 min, but not nuclear translocation of MAP kinase occurred after stimulation with EGF. The results suggest that sustained activation of the MAP kinase cascade may be required for MAP kinase to enter the nucleus, where it may initiate the gene transcription events required for neuronal differentiation of PC12 cells.

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Involvement of Ras/MAP Kinase in the Regulation of Ca2+ Channels in Adult Bullfrog Sympathetic Neurons by Nerve Growth Factor

Journal of Neurophysiology ◽

10.1152/jn.1998.80.3.1352 ◽

1998 ◽

Vol 80 (3) ◽

pp. 1352-1361 ◽

Cited By ~ 21

Author(s):

Saobo Lei ◽

William F. Dryden ◽

Peter A. Smith

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Protein Kinase ◽

Map Kinase ◽

Kinase Inhibitors ◽

Sympathetic Neurons ◽

Mitogen Activated Protein Kinase ◽

Nerve Growth ◽

Channel Current ◽

Mitogen Activated Protein

Lei, Saobo, William F. Dryden, and Peter A. Smith. Involvement of Ras/MAP kinase in the regulation of Ca2+ channels in adult bullfrog sympathetic neurons by nerve growth factor. J. Neurophysiol. 80: 1352–1361, 1998. The cellular mechanisms that underlie nerve growth factor (NGF) induced increase in Ca2+-channel current in adult bullfrog sympathetic B-neurons were examined by whole cell recording techniques. Cells were maintained at low density in neuron-enriched, defined-medium, serum-free tissue culture for 6 days in the presence or absence of NGF (200 ng/ml). The increase in Ba2+ current ( I Ba) density induced by NGF was attenuated by the RNA synthesis inhibitor cordycepin (20 μM), by the DNA transcription inhibitor actinomycin D (0.01 μg/ml), by inhibitors of Ras isoprenylation (perillic acid 0.1–1.0 mM or α-hydroxyfarnesylphosphonic acid 10–100 μM), by tyrosine kinase inhibitors genistein (20 μM) or lavendustin A (1 μM), and by PD98059 (10–100 μM), an inhibitor of mitogen-activated protein kinase kinase. Inhibitors of the phosphatidylinositol 3-kinase (PI3K) pathway (wortmannin, 100 nM, or LY29400, 100 μM) were ineffective as were inhibitors of phospholipase Cγ (U73122 or neomycin, both 100 μM). The effect of NGF persisted in Ca2+-free medium that contained 1.8 mM Mg2+ and 2 mM ethylene glycol-bis(β-aminoethyl ether)- N, N, N′, N′-tetraacetic acid. It was mimicked by a Trk antibody that was capable of inducing neurite outgrowth in explant cultures of bullfrog sympathetic ganglion. Antibodies raised against the low-affinity p75 neurotrophin receptor were ineffective in blocking the effect of NGF on I Ba. These results suggest that NGF-induced increase in Ca2+ channel current in adult sympathetic neurons results, at least in part, from new channel synthesis after Trk activation of Ras and mitogen activated protein kinase by a mechanism that is independent of extracellular Ca2+.

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A Novel Regulatory Mechanism in the Mitogen-activated Protein (MAP) Kinase Cascade

Journal of Biological Chemistry ◽

10.1074/jbc.272.51.32642 ◽

1997 ◽

Vol 272 (51) ◽

pp. 32642-32648 ◽

Cited By ~ 126

Author(s):

Makoto Fukuda ◽

Isamu Gotoh ◽

Makoto Adachi ◽

Yukiko Gotoh ◽

Eisuke Nishida

Keyword(s):

Map Kinase ◽

Regulatory Mechanism ◽

Map Kinase Cascade ◽

Protein Map ◽

Mitogen Activated Protein ◽

Kinase Cascade

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Regulation of cotton (Gossypium hirsutum ) drought responses by mitogen-activated protein (MAP) kinase cascade-mediated phosphorylation of GhWRKY59

New Phytologist ◽

10.1111/nph.14680 ◽

2017 ◽

Vol 215 (4) ◽

pp. 1462-1475 ◽

Cited By ~ 23

Author(s):

Fangjun Li ◽

Maoying Li ◽

Ping Wang ◽

Kevin L. Cox ◽

Liusheng Duan ◽

...

Keyword(s):

Gossypium Hirsutum ◽

Map Kinase ◽

Map Kinase Cascade ◽

Protein Map ◽

Mitogen Activated Protein ◽

Kinase Cascade

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The Stress-activated Mitogen-activated Protein Kinase Signaling Cascade Promotes Exit from Mitosis

Molecular Biology of the Cell ◽

10.1091/mbc.e05-12-1102 ◽

2006 ◽

Vol 17 (7) ◽

pp. 3136-3146 ◽

Cited By ~ 23

Author(s):

Vladimír Reiser ◽

Katharine E. D’Aquino ◽

Ly-Sha Ee ◽

Angelika Amon

Keyword(s):

Map Kinase ◽

Mitogen Activated Protein Kinase ◽

Hog Pathway ◽

Hypertonic Stress ◽

Map Kinase Cascade ◽

Protein Map ◽

Mitogen Activated Protein ◽

Early Anaphase ◽

Kinase Cascade ◽

Protein Kinase Signaling

In budding yeast, a signaling network known as the mitotic exit network (MEN) triggers exit from mitosis. We find that hypertonic stress allows MEN mutants to exit from mitosis in a manner dependent on the high osmolarity glycerol (HOG) mitogen-activated protein (MAP) kinase cascade. The HOG pathway drives exit from mitosis in MEN mutants by promoting the activation of the MEN effector, the protein phosphatase Cdc14. Activation of Cdc14 depends on the Cdc14 early anaphase release network, a group of proteins that functions in parallel to the MEN to promote Cdc14 function. Notably, exit from mitosis is promoted by the signaling branch defined by the Sho1 osmosensing system, but not by the Sln1 osmosensor of the HOG pathway. Our results suggest that the stress MAP kinase pathway mobilizes programs to promote completion of the cell cycle and entry into G1 under unfavorable conditions.

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Role of a Mitogen-Activated Protein Kinase Cascade in Ion Flux-Mediated Turgor Regulation in Fungi

Eukaryotic Cell ◽

10.1128/ec.5.3.480-487.2006 ◽

2006 ◽

Vol 5 (3) ◽

pp. 480-487 ◽

Cited By ~ 34

Author(s):

Roger R. Lew ◽

Natalia N. Levina ◽

Lana Shabala ◽

Marinela I. Anderca ◽

Sergey N. Shabala

Keyword(s):

Map Kinase ◽

Mitogen Activated Protein Kinase ◽

Wild Type ◽

Turgor Regulation ◽

Map Kinase Cascade ◽

Protein Map ◽

Mitogen Activated Protein ◽

Kinase Cascade ◽

Protein Kinase Cascade

ABSTRACT Fungi normally maintain a high internal hydrostatic pressure (turgor) of about 500 kPa. In response to hyperosmotic shock, there are immediate electrical changes: a transient depolarization (1 to 2 min) followed by a sustained hyperpolarization (5 to 10 min) prior to turgor recovery (10 to 60 min). Using ion-selective vibrating probes, we established that the transient depolarization is due to Ca2+ influx and the sustained hyperpolarization is due to H+ efflux by activation of the plasma membrane H+-ATPase. Protein synthesis is not required for H+-ATPase activation. Net K+ and Cl− uptake occurs at the same time as turgor recovery. The magnitude of the ion uptake is more than sufficient to account for the osmotic gradients required for turgor to return to its original level. Two osmotic mutants, os-1 and os-2, homologs of a two-component histidine kinase sensor and the yeast high osmotic glycerol mitogen-activated protein (MAP) kinase, respectively, have lower turgor than the wild type and do not exhibit the sustained hyperpolarization after hyperosmotic treatment. The os-1 mutant does not exhibit all of the wild-type turgor-adaptive ion fluxes (Cl− uptake increases, but net K+ flux barely changes and net H+ efflux declines) (os-2 was not examined). Both os mutants are able to regulate turgor but at a lower level than the wild type. Our results demonstrate that a MAP kinase cascade regulates ion transport, activation of the H+-ATPase, and net K+ and Cl− uptake during turgor regulation. Other pathways regulating turgor must also exist.

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Somatostatin Activation of Mitogen-Activated Protein Kinase via Somatostatin Receptor 1 (SSTR1)

Molecular Endocrinology ◽

10.1210/mend.13.1.0224 ◽

1999 ◽

Vol 13 (1) ◽

pp. 24-37 ◽

Cited By ~ 44

Author(s):

Tullio Florio ◽

Hong Yao ◽

Kendall D. Carey ◽

Tara J. Dillon ◽

Philip J. S. Stork

Keyword(s):

Growth Factor ◽

Protein Kinase ◽

Map Kinase ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Somatostatin Receptor ◽

Fibroblast Growth ◽

Map Kinase Cascade ◽

Mitogen Activated Protein ◽

Kinase Cascade

Abstract Hormones and growth factors regulate cell growth via the mitogen-activated protein (MAP) kinase cascade. Here we examine the actions of the hormone somatostatin on the MAP kinase cascade through one of its two major receptor subtypes, the somatostatin receptor 1 (SSTR1) stably expressed in CHO-K1 cells. Somatostatin antagonizes the proliferative effects of fibroblast growth factor in CHO-SSTR1 cells via the SSTR1 receptor. However, in these cells, somatostatin robustly activates MAP kinase (also called extracellular signal regulated kinase; ERK) and augments fibroblast growth factor-stimulated ERK activity. We show that the activation of ERK via SSTR1 is pertussis toxin sensitive and requires the small G protein Ras, phosphatidylinositol 3-kinase, the serine/threonine kinase Raf-1, and the protein tyrosine phosphatase SHP-2. The activation of ERK by SSTR1 increased the expression of the cyclin-dependent protein kinase inhibitor p21cip1/WAF1. Previous studies have suggested that somatostatin-stimulated protein tyrosine phosphatase activity mediates the growth effects of somatostatin. Our data suggest that SHP-2 stimulation by SSTR1 may mediate some of these effects through the activation of the MAP kinase cascade and the expression of p21cip1/WAF1.

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Genetic Analysis of rolled, Which Encodes a Drosophila Mitogen-Activated Protein Kinase

Genetics ◽

10.1093/genetics/153.2.763 ◽

1999 ◽

Vol 153 (2) ◽

pp. 763-771 ◽

Cited By ~ 1

Author(s):

Young-Mi Lim ◽

Kimiko Nishizawa ◽

Yoshimi Nishi ◽

Leo Tsuda ◽

Yoshihiro H Inoue ◽

...

Keyword(s):

Map Kinase ◽

Mitogen Activated Protein Kinase ◽

Female Sterility ◽

Tor Signaling ◽

Map Kinase Cascade ◽

Dominant Female ◽

Protein Map ◽

Mitogen Activated Protein ◽

Kinase Cascade

Abstract Genetic and molecular characterization of the dominant suppressors of D-rafC110 on the second chromosome identified two gain-of-function alleles of rolled (rl), which encodes a mitogen-activated protein (MAP) kinase in Drosophila. One of the alleles, rlSu23, was found to bear the same molecular lesion as rlSem, which has been reported to be dominant female sterile. However, rlSu23 and the current stock of rlSem showed only a weak dominant female sterility. Detailed analyses of the rl mutations demonstrated moderate dominant activities of these alleles in the Torso (Tor) signaling pathway, which explains the weak dominant female sterility observed in this study. The dominant rl mutations failed to suppress the terminal class maternal-effect mutations, suggesting that activation of Rl is essential, but not sufficient, for Tor signaling. Involvement of rl in cell proliferation was also demonstrated by clonal analysis. Branching and integration of signals in the MAP kinase cascade is discussed.

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