LETTERS 270 NATURE CELL BIOLOGY VOLUME 7 | NUMBER 3 | MARCH 2005 PAR-6–PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiam1 Takashi Nishimura 1 , Tomoya Yamaguchi 1 , Katsuhiro Kato 1 , Masato Yoshizawa 2 , Yo-ichi Nabeshima 2 , Shigeo Ohno 3 , Mikio Hoshino 2,4 and Kozo Kaibuchi 1,5 A polarity complex of PAR-3, PAR-6 and atypical protein kinase C (aPKC) functions in various cell-polarization events, including neuron specification 1–4 . The small GTPase Cdc42 binds to PAR- 6 and regulates cell polarity. However, little is known about the downstream signals of the Cdc42–PAR protein complex. Here, we found that PAR-3 directly interacted with STEF/Tiam1, which are Rac-specific guanine nucleotide-exchange factors, and that STEF formed a complex with PAR-3–aPKC–PAR- 6–Cdc42-GTP. Cdc42 induces lamellipodia in a Rac-dependent manner in N1E-115 neuroblastoma cells. Disruption of Cdc42– PAR-6 or PAR-3–STEF binding inhibited Cdc42-induced lamellipodia but not filopodia. The isolated STEF-binding PAR- 3 fragment was sufficient to induce lamellipodia independently of Cdc42 and PAR-6. PAR-3 is required for Cdc42-induced Rac activation, but is not essential for lamellipodia formation itself. In cultured hippocampal neurons, STEF accumulated at the tip of the growing axon and colocalized with PAR-3. The spatio-temporal activation and signalling of Cdc42–PAR-6–PAR- 3–STEF/Tiam1–Rac seem to be involved in neurite growth and axon specification. We propose that the PAR-6–PAR-3 complex mediates Cdc42-induced Rac activation by means of STEF/Tiam1, and that this process seems to be required for the establishment of neuronal polarity. The neuron is one of the most highly polarized cells, comprising two structurally and molecularly distinct parts — an axon and dendrites 5–7 . The PAR complex accumulates at the tip of the axon, and its polarized localiza- tion is important for axon specification 3,4 . Recently, we reported that PAR-3 is transported to the distal tip of neurites by the KIF3 (kinesin-2) motor 4 . The Rho family GTPases are crucial for the regulation of actin dynam- ics 8,9 . In neurons, Rho GTPases regulate growth-cone morphology, which affects the rate and direction of neurite growth and presumably results in axon specification 7 . Cdc42 and Rac1 are positive regulators of the growth cone through control of filopodia and lamellipodia formation, respectively. 1 Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa, Nagoya, Aichi 466-8550, Japan. 2 Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. 3 Department of Molecular Biology, Yokohama City University School of Medicine, Fuku-ura 3-9, Kanazawa-ku, Yokohama 236-0004, Japan. 4 PRESTO, JST, 4-1-8 Honcho Kawaguchi, Saitama, 332- 0012 Japan. 5 Correspondence should be addressed to K.K. (e-mail: kaibuchi@med.nagoya-u.ac.jp) Published online: 20 February 2005; DOI: 10.1038/ncb1227 Cdc42 is involved in polarity establishment in a wide variety of cell types, including neurons 10,11 . The localized activation of phosphatidylinositol-3- kinase (PI(3)K) and the accumulation of phosphoinositol-3,4,5-triphos- phate (PtdIns(3,4,5)P 3 ) are implicated in PAR-3 localization 3 and neuronal polarization 12 . PI(3)K regulates the activation of Cdc42 (ref. 9), and acti- vated Cdc42 directly binds to PAR-6 (refs 1,2) and activates aPKC 13,14 . These findings indicate that PI(3)K and Cdc42 function as upstream molecules of the PAR protein complex to regulate cell polarity. Tiam1 and STEF (also called Tiam2), which are Rac-specific guanine nucleotide-exchange factors (GEFs), can induce neurite-like processes in N1E-115 neuroblastoma cells 15,16 . The expression of a constitutively active Tiam1 leads to the development of multiple axon-like neurites in cultured hippocampal neurons 17 , indicating that the spatio-temporal activity of Rac is involved in axon specification. Although the activa- tion of Cdc42 leads to Rac activation 15,16,18 , the molecular mechanism by which this occurs and the link between this process and the establish- ment of cell polarity remain unknown. These observations prompted us to examine whether the Cdc42–PAR protein complex regulates neurite growth and neuronal polarity through an association with STEF/Tiam1 and Rac. We first examined the physiological association of the PAR complex with STEF/Tiam1 in a co-immunoprecipitation assay. When PAR-3 was immunoprecipitated from rat developing brain lysate, co- immunoprecipitation of STEF and Tiam1 was detected (Fig. 1a). PAR-3 was reciprocally co-immunoprecipitated with STEF or Tiam1. aPKC was also co-immunoprecipitated with STEF or Tiam1, indicating that STEF/Tiam1 associates with the PAR complex in vivo. We next examined whether the PAR complex directly binds to STEF. Endogenous STEF and Tiam1 were precipitated with glutathione-S- transferase (GST)–PAR-3-2N and GST–PAR-3-4N from rat brain lysate, but not with GST–PAR-6-WT (Fig. 1b, c). To examine which region of STEF is responsible for the interaction with PAR-3, affinity beads were incubated with COS7 cell lysate expressing various deletion fragments of STEF (Fig. 1d). STEF-full, STEF-∆N and STEF-PHnTSS interacted with GST–PAR-3-2N and GST–PAR-3-4N, whereas STEF-PDZ and Nature Publishing Group ©2005