Intact Ras function is required for sustained activation and nuclear translocation of extracellular signal-regulated kinases in nerve growth factor-stimulated 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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Sustained activation of M-Ras induced by nerve growth factor is essential for neuronal differentiation of PC12 cells

Genes to Cells ◽

10.1111/j.1365-2443.2006.01002.x ◽

2006 ◽

Vol 11 (9) ◽

pp. 1097-1113 ◽

Cited By ~ 36

Author(s):

Peng Sun ◽

Haruko Watanabe ◽

Kazunori Takano ◽

Takashi Yokoyama ◽

Jun-ichi Fujisawa ◽

...

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Neuronal Differentiation ◽

Pc12 Cells ◽

Nerve Growth ◽

Sustained Activation

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Nerve growth factor in combination with second messenger analogues causes neuronal differentiation of PC12 cells expressing a dominant inhibitory Ras protein without inducing activation of extracellular signal-regulated kinases

European Journal of Neuroscience ◽

10.1046/j.0953-816x.2001.01787.x ◽

2001 ◽

Vol 14 (9) ◽

pp. 1445-1454 ◽

Cited By ~ 10

Author(s):

Gábor Boglári ◽

József Szeberényi

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Neuronal Differentiation ◽

Pc12 Cells ◽

Second Messenger ◽

Nerve Growth ◽

Ras Protein ◽

Extracellular Signal

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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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