Abstract
Neurite outgrowth is an important process in the formation of neuronal networks. Rac1 and Cdc42, members of the Rho-family GTPases, positively regulate neurite extension through reorganization of the actin cytoskeleton. Here, we examine the dynamic linkage between Rac1/Cdc42 and phosphatidylinositol 3-kinase (PI3-kinase) during nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Activity imaging using fluorescence resonance energy transfer probes showed that PI3-kinase as well as Rac1/Cdc42 was transiently activated in broad areas of the cell periphery immediately after NGF addition. Subsequently, local and repetitive activation of PI3-kinase and Rac1/Cdc42 was observed at the protruding sites. Depletion of Vav2 and Vav3 by RNA interference significantly inhibited both Rac1/Cdc42 activation and the formation of short processes leading to neurite outgrowth. At the NGF-induced protrusions, local phosphatidylinositol 3,4,5-trisphosphate accumulation recruited Vav2 and Vav3 to activate Rac1 and Cdc42, and conversely, Vav2 and Vav3 were required for the local activation of PI3-kinase. These observations demonstrated for the first time that Vav2 and Vav3 are essential constituents of the positive feedback loop that is comprised of PI3-kinase and Rac1/Cdc42 and cycles locally with morphological changes.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Base Sequence
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Biological Transport, Active / drug effects
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Cell Cycle Proteins / antagonists & inhibitors
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cell Membrane / drug effects
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Cell Membrane / metabolism
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Feedback
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Fluorescence Resonance Energy Transfer
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Nerve Growth Factor / pharmacology
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Neurites / drug effects
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Neurites / metabolism*
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Neurites / ultrastructure*
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Neurons / drug effects
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Neurons / metabolism
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Neurons / ultrastructure
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Oncogene Proteins / antagonists & inhibitors
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Oncogene Proteins / genetics
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Oncogene Proteins / metabolism*
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PC12 Cells
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Phosphatidylinositol 3-Kinases / metabolism
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Phosphatidylinositol Phosphates / metabolism*
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Proto-Oncogene Proteins / antagonists & inhibitors
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism*
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Proto-Oncogene Proteins c-vav / antagonists & inhibitors
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Proto-Oncogene Proteins c-vav / genetics
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Proto-Oncogene Proteins c-vav / metabolism*
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RNA Interference
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RNA, Messenger / genetics
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RNA, Messenger / metabolism
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Rats
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SOS1 Protein / metabolism
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Signal Transduction
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Son of Sevenless Proteins / metabolism
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cdc42 GTP-Binding Protein / metabolism*
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rac1 GTP-Binding Protein / metabolism*
Substances
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Cell Cycle Proteins
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Oncogene Proteins
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Phosphatidylinositol Phosphates
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-vav
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RNA, Messenger
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SOS1 Protein
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Son of Sevenless Proteins
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VAV3 protein, rat
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Vav1 protein, rat
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Vav2 protein, rat
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phosphatidylinositol 3,4,5-triphosphate
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Nerve Growth Factor
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Phosphatidylinositol 3-Kinases
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cdc42 GTP-Binding Protein
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rac1 GTP-Binding Protein