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.Neurotrophins have long been recognized for their role in regulating neuronal survival, cell growth, differentiation, and neuronal plasticity. The archetypal neurotrophin, nerve growth factor (NGF), elicits most of these effects by binding and activating the receptor tyrosine kinase (RTK), TrkA, which leads to the activation of several well-defined signaling cascades. Of these, the phosphoinositide 3-kinase (PI3-K) and extracellular signal-regulated kinase (ERK) pathways are two of the most extensively studied. PI3-Ks have been implicated in multiple biological responses including membrane trafficking, proliferation, differentiation, and survival (67). These kinases consist of a family of proteins which phosphorylate phosphatidylinositol (PI) at the D3 position and have been categorized into three classes based on their lipid substrate specificity in vitro (96). The lipid products of PI3-Ks {PI 3-phosphate [PI(3)P], PI(3,4)P, PI(3,5)P, and PI(3,4,5)P} are known to act as second messengers and mediate most of the known functions of PI3-Ks in cells (45).The mitogen-activated protein kinase family members, ERK1 and ERK2, can also be activated by a wide variety of stimuli to promote a diverse array of cellular functions (77). In addition to their established rol...

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