Role of Phosphoinositide 3-Kinase and Endocytosis in Nerve Growth Factor-Induced Extracellular Signal-Regulated Kinase Activation via Ras and Rap1

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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Nerve Growth Factor Activates Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Pathways To Stimulate CREB Serine 133 Phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.18.4.1946 ◽

1998 ◽

Vol 18 (4) ◽

pp. 1946-1955 ◽

Cited By ~ 350

Author(s):

Jun Xing ◽

Jon M. Kornhauser ◽

Zhengui Xia ◽

Elizabeth A. Thiele ◽

Michael E. Greenberg

Keyword(s):

Transcription Factor ◽

Nerve Growth Factor ◽

Growth Factor ◽

Protein Kinase ◽

Mitogen Activated Protein Kinase ◽

Nerve Growth ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal ◽

Mitogen Activated Protein ◽

Ribosomal S6 Kinase

ABSTRACT The mechanisms by which growth factor-induced signals are propagated to the nucleus, leading to the activation of the transcription factor CREB, have been characterized. Nerve growth factor (NGF) was found to activate multiple signaling pathways that mediate the phosphorylation of CREB at the critical regulatory site, serine 133 (Ser-133). NGF activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs), which in turn activate the pp90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases, all three members of which were found to catalyze CREB Ser-133 phosphorylation in vitro and in vivo. In addition to the ERK/RSK pathway, we found that NGF activated the p38 MAPK and its downstream effector, MAPK-activated protein kinase 2 (MAPKAP kinase 2), resulting in phosphorylation of CREB at Ser-133. Inhibition of either the ERK/RSK or the p38/MAPKAP kinase 2 pathway only partially blocked NGF-induced CREB Ser-133 phosphorylation, suggesting that either pathway alone is sufficient for coupling the NGF signal to CREB activation. However, inhibition of both the ERK/RSK and the p38/MAPKAP kinase 2 pathways completely abolished NGF-induced CREB Ser-133 phosphorylation. These findings indicate that NGF activates two distinct MAPK pathways, both of which contribute to the phosphorylation of the transcription factor CREB and the activation of immediate-early genes.

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Sustained activation of extracellular signal-regulated kinase by nerve growth factor regulates c-fos protein stabilization and transactivation in PC12 cells

Journal of Neurochemistry ◽

10.1111/j.1471-4159.2006.04250.x ◽

2006 ◽

Vol 99 (6) ◽

pp. 1480-1493 ◽

Cited By ~ 37

Author(s):

Michael J. Pellegrino ◽

Philip J. S. Stork

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Pc12 Cells ◽

Protein Stabilization ◽

Nerve Growth ◽

Extracellular Signal Regulated Kinase ◽

Fos Protein ◽

Extracellular Signal ◽

Sustained Activation

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Nerve Growth Factor Stimulates the Concentration of TrkA within Lipid Rafts and Extracellular Signal-Regulated Kinase Activation through c-Cbl-Associated Protein

Molecular and Cellular Biology ◽

10.1128/mcb.01109-06 ◽

2007 ◽

Vol 27 (16) ◽

pp. 5686-5698 ◽

Cited By ~ 48

Author(s):

Allison S. Limpert ◽

J. Colleen Karlo ◽

Gary E. Landreth

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Neuronal Differentiation ◽

Lipid Rafts ◽

Dorsal Root Ganglion Neurons ◽

Membrane Microdomains ◽

Nerve Growth ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal ◽

Ganglion Neurons

ABSTRACT Nerve growth factor (NGF) acts through its receptor, TrkA, to elicit the neuronal differentiation of PC12 cells through the action of extracellular signal-regulated kinase 1 (ERK1) and ERK2. Upon NGF binding, TrkA translocates and concentrates in cholesterol-rich membrane microdomains or lipid rafts, facilitating formation of receptor-associated signaling complexes, activation of downstream signaling pathways, and internalization into endosomes. We have investigated the mechanisms responsible for the localization of TrkA within lipid rafts and its ability to activate ERK1 and ERK2. We report that NGF treatment results in the translocation of activated forms of TrkA to lipid rafts, and this localization is important for efficient activation of the ERKs. TrkA is recruited and retained within lipid rafts through its association with flotillin, an intrinsic constituent of these membrane microdomains, via the adapter protein, c-Cbl associated protein (CAP). Mutant forms of CAP that lack protein interaction domains block TrkA localization to lipid rafts and attenuate ERK activation. Importantly, suppression of endogenous CAP expression inhibited NGF-stimulated neurite outgrowth from primary dorsal root ganglion neurons. These data provide a mechanism for the lipid raft localization of TrkA and establish the importance of the CAP adaptor protein for NGF activation of the ERKs and neuronal differentiation.

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Nerve Growth Factor Activation of the Extracellular Signal-Regulated Kinase Pathway Is Modulated by Ca2+and Calmodulin

Molecular and Cellular Biology ◽

10.1128/mcb.20.6.1931-1946.2000 ◽

2000 ◽

Vol 20 (6) ◽

pp. 1931-1946 ◽

Cited By ~ 33

Author(s):

Joaquim Egea ◽

Carme Espinet ◽

Rosa M. Soler ◽

Sandra Peiró ◽

Nativitat Rocamora ◽

...

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Pc12 Cells ◽

Sympathetic Neurons ◽

Moderate Increase ◽

Dependent Manner ◽

Nerve Growth ◽

Extracellular Signal Regulated Kinase ◽

Extracellular Signal ◽

Kinase Pathway

ABSTRACT Nerve growth factor is a member of the neurotrophin family of trophic factors that have been reported to be essential for the survival and development of sympathetic neurons and a subset of sensory neurons. Nerve growth factor exerts its effects mainly by interaction with the specific receptor TrkA, which leads to the activation of several intracellular signaling pathways. Once activated, TrkA also allows for a rapid and moderate increase in intracellular calcium levels, which would contribute to the effects triggered by nerve growth factor in neurons. In this report, we analyzed the relationship of calcium to the activation of the Ras/extracellular signal-regulated kinase pathway in PC12 cells. We observed that calcium and calmodulin are both necessary for the acute activation of extracellular signal-regulated kinases after TrkA stimulation. We analyzed the elements of the pathway that lead to this activation, and we observed that calmodulin antagonists completely block the initial Raf-1 activation without affecting the function of upstream elements, such as Ras, Grb2, Shc, and Trk. We have broadened our study to other stimuli that activate extracellular signal-regulated kinases through tyrosine kinase receptors, and we have observed that calmodulin also modulates the activation of such kinases after epidermal growth factor receptor stimulation in PC12 cells and after TrkB stimulation in cultured chicken embryo motoneurons. Calmodulin seems to regulate the full activation of Raf-1 after Ras activation, since functional Ras is necessary for Raf-1 activation after nerve growth factor stimulation and calmodulin-Sepharose is able to precipitate Raf-1 in a calcium-dependent manner.

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