Phospholipase Activity of Phospholipase C-γ1 Is Required for Nerve Growth Factor-regulated MAP Kinase Signaling Cascade in PC12 Cells

ABSTRACT Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.

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Src Homology Domains of Phospholipase C γ1 Inhibit Nerve Growth Factor-Induced Differentiation of PC12 Cells

Journal of Neurochemistry ◽

10.1046/j.1471-4159.1998.71010178.x ◽

2002 ◽

Vol 71 (1) ◽

pp. 178-185 ◽

Cited By ~ 29

Author(s):

Sun Sik Bae ◽

Young Han Lee ◽

Jong-Soo Chang ◽

Sehamuddin H. Galadari ◽

Yong Sik Kim ◽

...

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Phospholipase C ◽

Pc12 Cells ◽

Induced Differentiation ◽

Nerve Growth ◽

Src Homology ◽

Src Homology Domains ◽

Homology Domains

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Pertussis toxin-sensitive Gi/o proteins are involved in nerve growth factor-induced pro-survival Akt signaling cascade in PC12 cells

Cellular Signalling ◽

10.1016/j.cellsig.2004.11.008 ◽

2005 ◽

Vol 17 (7) ◽

pp. 881-890 ◽

Cited By ~ 26

Author(s):

Eddy H.T. Wu ◽

Yung H. Wong

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Pc12 Cells ◽

Pertussis Toxin ◽

Akt Signaling ◽

Signaling Cascade ◽

Nerve Growth

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Nitric Oxide Synthase Inhibitors Modulate Nerve-Growth-Factor Mediated Activation of Akt

ISRN Cell Biology ◽

10.5402/2012/847974 ◽

2012 ◽

Vol 2012 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Cheryl L. Cragg ◽

Janet C. MacKinnon ◽

Bettina E. Kalisch

Keyword(s):

Nitric Oxide ◽

Nerve Growth Factor ◽

Growth Factor ◽

Map Kinase ◽

Pc12 Cells ◽

Cell Lines ◽

Nerve Growth ◽

Akt Phosphorylation ◽

Map Kinase Pathway ◽

Kinase Pathway

Nitric oxide (NO) modulates nerve-growth-factor- (NGF-) mediated signaling and gene expression. In the present paper, the role of NO in NGF-mediated Akt activation in PC12 and IMR32 cells was investigated. Cells were treated with NGF (50 ng/mL) in the presence or absence of NO synthase (NOS) inhibitors and Akt phosphorylation assessed by western blot analysis. In both cell lines, Akt was phosphorylated within 15 min of NGF treatment. In PC12 cells, this level of phosphorylation was sustained for 60 min, while in IMR32 cells, the activation decreased after 30 min of NGF treatment. The nonselective NOS inhibitor Nω-nitro-L-arginine methylester (L-NAME; 20 mM) had no effect on NGF-mediated Akt phosphorylation in PC12 cells but in combination with NGF, the iNOS selective inhibitor s-methylisothiourea (S-MIU; 2.0 mM) maintained Akt phosphorylation up to 2 h. In IMR32 cells, both L-NAME and S-MIU prolonged the activation of Akt. Pretreatment with 50 μM U0126, a MAP kinase pathway inhibitor, also increased the activation of Akt in both cell lines. These data suggest that NO modulates the duration of phosphorylation of Akt in response to NGF and that this effect may, in part, be mediated by the effects of NO on the Ras-MAP kinase pathway.

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Essential Role for Phospholipase D2 Activation Downstream of ERK MAP Kinase in Nerve Growth Factor-stimulated Neurite Outgrowth from PC12 Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m402610200 ◽

2004 ◽

Vol 279 (36) ◽

pp. 37870-37877 ◽

Cited By ~ 37

Author(s):

Hiroshi Watanabe ◽

Takeaki Yokozeki ◽

Masakazu Yamazaki ◽

Hideyuki Miyazaki ◽

Takehiko Sasaki ◽

...

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Map Kinase ◽

Neurite Outgrowth ◽

Pc12 Cells ◽

Essential Role ◽

Nerve Growth ◽

Phospholipase D2

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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.14.10.6944-6953.1994 ◽

1994 ◽

Vol 14 (10) ◽

pp. 6944-6953

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 Signaling by Phospholipase C-γ Mediates Nerve Growth Factor-Triggered Gene Expression

Molecular and Cellular Biology ◽

10.1128/mcb.21.8.2695-2705.2001 ◽

2001 ◽

Vol 21 (8) ◽

pp. 2695-2705 ◽

Cited By ~ 36

Author(s):

Deog-Young Choi ◽

Juan Jose Toledo-Aral ◽

Rosalind Segal ◽

Simon Halegoua

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Binding Site ◽

Phospholipase C ◽

Pc12 Cells ◽

Nerve Growth ◽

Neuronal Gene ◽

Intracellular Ca ◽

Kinetics Of

ABSTRACT In contrast to conventional signaling by growth factors that requires their continual presence, a 1-min pulse of nerve growth factor (NGF) is sufficient to induce electrical excitability in PC12 cells due to induction of the peripheral nerve type 1 (PN1) sodium channel gene. We have investigated the mechanism for this triggered signaling pathway by NGF in PC12 cells. Mutation of TrkA at key autophosphorylation sites indicates an essential role for the phospholipase C-γ (PLC-γ) binding site, but not the Shc binding site, for NGF-triggered induction of PN1. In concordance with results with Trk mutants, drug-mediated inhibition of PLC-γ activity also blocks PN1 induction by NGF. Examination of the kinetics of TrkA autophosphorylation indicates that triggered signaling does not result from sustained activation and autophosphorylation of the TrkA receptor kinase, whose phosphorylation state declines rapidly after NGF removal. Rather, TrkA triggers an unexpectedly prolonged phosphorylation and activation of PLC-γ signaling that is sustained for up to 2 h. Prevention of the elevation of intracellular Ca2+ levels using BAPTA-AM results in a block of PN1 induction by NGF. Sustained signaling by PLC-γ provides a means for differential neuronal gene induction after transient exposure to NGF.

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