Local phosphatidylinositol 3,4,5-trisphosphate accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells.

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.

[1]  L. Greene,et al.  Neurite outgrowth in peripherin-depleted PC12 cells , 1992, The Journal of cell biology.

[2]  K. Schuebel,et al.  Isolation and characterization of murine vav2, a member of the vav family of proto-oncogenes. , 1996, Oncogene.

[3]  John G. Collard,et al.  The Guanine Nucleotide Exchange Factor Tiam1 Affects Neuronal Morphology; Opposing Roles for the Small GTPases Rac and Rho , 1997, The Journal of cell biology.

[4]  L. Van Aelst,et al.  Rho GTPases and signaling networks. , 1997, Genes & development.

[5]  K. Schuebel,et al.  Phosphotyrosine-dependent activation of Rac-1 GDP/GTP exchange by the vav proto-oncogene product , 1997, Nature.

[6]  A. Wong,et al.  Grb2-associated binder-1 mediates phosphatidylinositol 3-kinase activation and the promotion of cell survival by nerve growth factor. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[7]  H. Iba,et al.  Expression of a Constitutively Active Phosphatidylinositol 3-Kinase Induces Process Formation in Rat PC12 Cells , 1997, The Journal of Biological Chemistry.

[8]  C. Der,et al.  Lck regulates Vav activation of members of the Rho family of GTPases , 1997, Molecular and cellular biology.

[9]  D. Bar-Sagi,et al.  Coupling of Ras and Rac guanosine triphosphatases through the Ras exchanger Sos. , 1998, Science.

[10]  M. White,et al.  Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. , 1998, Science.

[11]  Gary G. Borisy,et al.  Arp2/3 Complex and Actin Depolymerizing Factor/Cofilin in Dendritic Organization and Treadmilling of Actin Filament Array in Lamellipodia , 1999, The Journal of cell biology.

[12]  B. Mueller,et al.  Growth cone guidance: first steps towards a deeper understanding. , 1999, Annual review of neuroscience.

[13]  C. Betsholtz,et al.  EPS8 and E3B1 transduce signals from Ras to Rac , 1999, Nature.

[14]  X. Bustelo,et al.  Biological and Regulatory Properties of Vav-3, a New Member of the Vav Family of Oncoproteins , 1999, Molecular and Cellular Biology.

[15]  D. Ginty,et al.  Spatially and Functionally Distinct Roles of the PI3-K Effector Pathway during NGF Signaling in Sympathetic Neurons , 2000, Neuron.

[16]  Frederick W. Alt,et al.  Vav Family Proteins Couple to Diverse Cell Surface Receptors , 2000, Molecular and Cellular Biology.

[17]  K. Burridge,et al.  Vav2 Activates Rac1, Cdc42, and RhoA Downstream from Growth Factor Receptors but Not β1 Integrins , 2000, Molecular and Cellular Biology.

[18]  T. Weissman,et al.  Identification of diverse nerve growth factor-regulated genes by serial analysis of gene expression (SAGE) profiling. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[19]  L. Luo.  RHO GTPASES in neuronal morphogenesis , 2000, Nature Reviews Neuroscience.

[20]  D Broek,et al.  Control of Intramolecular Interactions between the Pleckstrin Homology and Dbl Homology Domains of Vav and Sos1 Regulates Rac Binding* , 2000, The Journal of Biological Chemistry.

[21]  David R Kaplan,et al.  Neurotrophin signal transduction in the nervous system , 2000, Current Opinion in Neurobiology.

[22]  S. O’Brien,et al.  Skeletal muscle deformity and neuronal disorder in Trio exchange factor-deficient mouse embryos. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Y. Barde,et al.  Neurotrophins: key regulators of cell fate and cell shape in the vertebrate nervous system. , 2000, Genes & development.

[24]  Zhenbiao Yang,et al.  RHO Gtpases and the Actin Cytoskeleton , 2000 .

[25]  Y. Yamaguchi,et al.  Small GTPase RhoG Is a Key Regulator for Neurite Outgrowth in PC12 Cells , 2000, Molecular and Cellular Biology.

[26]  M. Ichihashi,et al.  Involvement of an SHP-2-Rho small G protein pathway in hepatocyte growth factor/scatter factor-induced cell scattering. , 2000, Molecular biology of the cell.

[27]  X. Bustelo.  Regulatory and Signaling Properties of the Vav Family , 2000, Molecular and Cellular Biology.

[28]  M. Greenberg,et al.  EphA Receptors Regulate Growth Cone Dynamics through the Novel Guanine Nucleotide Exchange Factor Ephexin , 2001, Cell.

[29]  X. Bustelo.  Vav proteins, adaptors and cell signaling , 2001, Oncogene.

[30]  A. Miyawaki,et al.  Spatio-temporal images of growth-factor-induced activation of Ras and Rap1 , 2001, Nature.

[31]  K. Mori,et al.  Differential Responses to Nerve Growth Factor and Epidermal Growth Factor in Neurite Outgrowth of PC12 Cells Are Determined by Rac1 Activation Systems* , 2001, The Journal of Biological Chemistry.

[32]  P. Marignani,et al.  Vav2 is required for cell spreading , 2001, The Journal of cell biology.

[33]  E. Nishida,et al.  ERK induces p35, a neuron-specific activator of Cdk5, through induction of Egr1 , 2001, Nature Cell Biology.

[34]  K. Kosik,et al.  Evidence for the Involvement of Tiam1 in Axon Formation , 2001, The Journal of Neuroscience.

[35]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[36]  A. Markus,et al.  Neurotrophic factors and axonal growth , 2002, Current Opinion in Neurobiology.

[37]  S. Diriong,et al.  The Human Rho-GEF Trio and Its Target GTPase RhoG Are Involved in the NGF Pathway, Leading to Neurite Outgrowth , 2002, Current Biology.

[38]  E. Feldman,et al.  Signaling mechanisms that regulate actin‐based motility processes in the nervous system , 2002, Journal of neurochemistry.

[39]  D. Bar-Sagi,et al.  The Two Hats of SOS , 2002, Science's STKE.

[40]  E. Gosmanova,et al.  Nerve Growth Factor Signals through TrkA, Phosphatidylinositol 3-Kinase, and Rac1 to Inactivate RhoA during the Initiation of Neuronal Differentiation of PC12 Cells* , 2002, The Journal of Biological Chemistry.

[41]  D. Kioussis,et al.  Vav1 Transduces T Cell Receptor Signals to the Activation of Phospholipase C-γ1 via Phosphoinositide 3-Kinase-dependent and -independent Pathways , 2002, The Journal of experimental medicine.

[42]  L. Wang,et al.  Distinct Role of Phosphatidylinositol 3-Kinase and Rho Family GTPases in Vav3-induced Cell Transformation, Cell Motility, and Morphological Changes* , 2002, The Journal of Biological Chemistry.

[43]  H. Bourne,et al.  A chemical compass. , 2002, Nature.

[44]  Takeshi Nakamura,et al.  Grit, a GTPase-Activating Protein for the Rho Family, Regulates Neurite Extension through Association with the TrkA Receptor and N-Shc and CrkL/Crk Adapter Molecules , 2002, Molecular and Cellular Biology.

[45]  R. Bernards,et al.  A System for Stable Expression of Short Interfering RNAs in Mammalian Cells , 2002, Science.

[46]  Marc W. Kirschner,et al.  A PtdInsP3- and Rho GTPase-mediated positive feedback loop regulates neutrophil polarity , 2002, Nature Cell Biology.

[47]  A. Hall,et al.  Guanine nucleotide exchange factors for Rho GTPases: turning on the switch. , 2002, Genes & development.

[48]  J. Downward,et al.  Vav3 Modulates B Cell Receptor Responses by Regulating Phosphoinositide 3-Kinase Activation , 2002, The Journal of experimental medicine.

[49]  M. Matsuda,et al.  Activation of Rac and Cdc42 Video Imaged by Fluorescent Resonance Energy Transfer-Based Single-Molecule Probes in the Membrane of Living Cells , 2002, Molecular and Cellular Biology.

[50]  Lewis C Cantley,et al.  The phosphoinositide 3-kinase pathway. , 2002, Science.

[51]  Paul Herzmark,et al.  Lipid products of PI(3)Ks maintain persistent cell polarity and directed motility in neutrophils , 2002, Nature Cell Biology.

[52]  P. Devreotes,et al.  Temporal and spatial regulation of chemotaxis. , 2002, Developmental cell.

[53]  Y. Jan,et al.  Hippocampal Neuronal Polarity Specified by Spatially Localized mPar3/mPar6 and PI 3-Kinase Activity , 2003, Cell.

[54]  Richard A. Firtel,et al.  Leading the way: directional sensing through phosphatidylinositol 3-kinase and other signaling pathways , 2003, Journal of Cell Science.

[55]  M. Matsuda,et al.  Mechanism of the spatio‐temporal regulation of Ras and Rap1 , 2003, The EMBO journal.

[56]  M. Matsuda,et al.  Activity of Rho-family GTPases during cell division as visualized with FRET-based probes , 2003, The Journal of cell biology.

[57]  Y. Umezawa,et al.  Production of PtdInsP3 at endomembranes is triggered by receptor endocytosis , 2003, Nature Cell Biology.

[58]  Daniel Kalman,et al.  Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis , 2003, The Journal of cell biology.

[59]  J. Downward,et al.  Mechanism of Epidermal Growth Factor Regulation of Vav2, a Guanine Nucleotide Exchange Factor for Rac* , 2003, The Journal of Biological Chemistry.

[60]  K. Aoki,et al.  Spatio-temporal Regulation of Rac1 and Cdc42 Activity during Nerve Growth Factor-induced Neurite Outgrowth in PC12 Cells* , 2004, Journal of Biological Chemistry.

[61]  E. Lam,et al.  Vav-Dependent and Vav-Independent Phosphatidylinositol 3-Kinase Activation in Murine B Cells Determined by the Nature of the Stimulus1 , 2004, The Journal of Immunology.

[62]  A. Alcover,et al.  Vav proteins, masters of the world of cytoskeleton organization. , 2004, Cellular signalling.