Here we identify a 65 kDa protein (N‐WASP) from brain that binds the SH3 domains of Ash/Grb2. The sequence is homologous to Wiskott‐Aldrich syndrome protein (WASP). N‐WASP has several functional motifs, such as a pleckstrin homology (PH) domain and cofilin‐homologous region, through which N‐WASP depolymerizes actin filaments. When overexpressed in COS 7 cells, the wild‐type N‐WASP causes several surface protrusions where N‐WASP co‐localizes with actin filaments. Epidermal growth factor (EGF) treatment induces the complex formation of EGF receptors and N‐WASP, and produces microspikes. On the other hand, two mutants, C38W (a point mutation in the PH domain) and deltaVCA (deletion of the actin binding domain), localize predominantly in the nucleus and do not cause a change in the cytoskeleton, irrespective of EGF treatment. Interestingly, the C38W PH domain binds less effectively to phosphatidylinositol 4,5‐bisphosphate (PIP2) than the wild‐type PH domain. These results suggest the importance of the PIP2 binding ability of the PH domain and the actin binding for retention in membranes. Collectively, we conclude that N‐WASP transmits signals from tyrosine kinases to cause a polarized rearrangement of cortical actin filaments dependent on PIP2.

Arf family GTP-binding proteins are best characterized as regulators of membrane traffic, but recent studies indicate an additional role in cytoskeletal organization. An Arf GTPase-activating protein of the centaurin ␤ family, ASAP1 (also known as centaurin ␤4), binds Arf and two other known regulators of the actin cytoskeleton, the tyrosine kinase Src and phosphatidylinositol 4,5-bisphosphate. In this paper, we show that ASAP1 localizes to focal adhesions and cycles with focal adhesion proteins when cells are stimulated to move. Overexpression of ASAP1 altered the morphology of focal adhesions and blocked both cell spreading and formation of dorsal ruffles induced by platelet-derived growth factor (PDGF). On the other hand, ASAP1, with a mutation that disrupted GTPase-activating protein activity, had a reduced effect on cell spreading and increased the number of cells forming dorsal ruffles in response to PDGF. These data support a role for an Arf GTPase-activating protein, ASAP1, as a regulator of cytoskeletal remodeling and raise the possibility that the Arf pathway is a target for PDGF signaling.

Nerve growth factor (NGF) stimulation of pheochromocytoma PC12 cells transiently increased the intracellular concentration of reactive oxygen species (ROS).This increase was blocked by the chemical antioxidant N-acetylcysteine and a flavoprotein inhibitor, diphenylene iodonium. NGF responses of PC12 cells, including neurite outgrowth, tyrosine phosphorylation, and AP-1 activation, was inhibited when ROS production was prevented by N-acetylcysteine and diphenylene iodonium. The expression of dominant negative Rac1N17 blocked induction of both ROS generation and morphological differentiation by NGF. The ROS produced appears to be H 2 O 2 , because the introduction of catalase into the cells abolished NGF-induced neurite outgrowth, ROS production, and tyrosine phosphorylation. These results suggest that the ROS, perhaps H 2 O 2 , acts as an intracellular signal mediator for NGF-induced neuronal differentiation and that NGF-stimulated ROS production is regulated by Rac1 and a flavoprotein-binding protein similar to the phagocytic NADPH oxidase.Reactive oxygen species (ROS) 1 that cause oxidative stress have generally been viewed as cytotoxic depending on the dose (1, 2). ROS are responsible for the host defense mechanism in neutrophils (3) and possess carcinogenic potential associated with tumor promotion (4, 5). Recent studies, however, indicate that small nontoxic amounts of ROS may play a normal role as a second messenger in the various signaling pathways (1). The production of ROS such as superoxide (O 2 . ) and hydrogen peroxide (H 2 O 2 ) was observed in a number of cells stimulated with cytokines such as transforming growth factors-␤1 (6, 7), interleukin-1 (8), and tumor necrosis factor ␣ (9) or peptide growth factors such as platelet-derived growth factor (PDGF) (10) and epidermal growth factor (EGF) (11). H 2 O 2 has been shown to mediate PDGF-induced cellular DNA synthesis of rat vascular smooth muscle cells (10). Ras-dependent cell growth requires generation of the O 2 . free radical through a pathway involving Rac1 (12). Although the role of ROS has been extensively studied in mitogenesis, inflammation, and apoptosis (1), little is known about its functional role in the differentiation process. The differentiation process in the nervous system is regulated by the action of differentiation and growth factors including NGF. NGF induces the growth arrest of PC12 cells and promotes their differentiation into sympathetic neuron-like cells (13). NGF binding to its receptor tyrosine kinase, TrkA, initiates various molecular interactions including tyrosine phosphorylation of proteins and the action of the Ras/Raf/MEK/MAPK pathway (14,15). NGF induces the production of reactive nitric oxide (NO), and NO is required for NGF-induced cytostasis and differentiation (16), suggesting that free radical molecules such as NO and ROS may exert a regulatory role in certain types of cellular differentiation. In the current study, we focused on the role of ROS and a small GTP-binding protein, Rac1, in the NGF-induced neuron...