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Vol. 16, Issue 5, 2207-2217, May 2005
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* Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan;
Department of Biochemistry, Hokkaido University School of Medicine, Hokkaido 060-8638, Japan
Submitted October 18, 2004;
Revised January 27, 2005;
Accepted February 10, 2005
Monitoring Editor: Anne Ridley
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.
Abbreviations used: BSA, bovine serum albumin; CRIB, Cdc42/Rac1 interacting binding; CFP, cyan fluorescent protein; DIC, differential interference contrast; FRET, fluorescence resonance energy transfer; GEF, guanine nucleotide exchange factor; IMD, intensity-modulated display; NGF, nerve growth factor; PBS, phosphate-buffered saline; PI3-kinase, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; RNAi, RNA interference; shRNA, short-hairpin RNA; YFP, yellow fluorescent protein.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).
Address correspondence to: Takeshi Nakamura (t-nakamu{at}biken.osaka-u.ac.jp).
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